Transgender and gender-nonconforming students at a Mississippi high school were unfairly targeted because of the school district’s sex-specific dress code, the American Civil Liberties Union claims in a complaint filed Wednesday with the U.S. Department of Education.

The rights advocacy organization, which brought the complaint on behalf of the mother of a transgender student, said the Harrison County School District’s dress code policy violates Title IX, a 1972 federal law which prohibits schools and colleges that accept federal funds from discriminating against people on the basis of sex.

The Mississippi school’s dress code policy for the 2023-2024 school year states that students must adhere to the “dress attire consistent with their biological sex” and that boys must wear shorts or pants and girls must wear dresses or skirts.

In practice, this policy has had harmful and humiliating consequences for transgender girls and gender-nonconforming cisgender girls alike, the complaint alleges.

In March, school administrators at Harrison Central High School blocked a 16-year-old transgender girl, identified as A.H. in the complaint, from wearing a dress at a regional band concert. School principal Kelly Fuller told A.H. that she could not “represent our school dressed like that” and gave her the choice to change into “boys’ clothes” or be sent to in-school suspension and forgo participating in the concert.

The ACLU also asserted that A.H. had been harassed while using the girls’ restroom and that when she tried to use the boys’ restroom as a last resort, she was screamed at by a teacher.

“I’m deeply concerned about the discriminatory practices within Harrison County School District that have unfairly targeted my daughter, along with other students,” said Kimberly Hudson, the mother of A.H., in a statement. “Transgender and gender nonconforming students should not be forced to choose between participating in school events or remaining true to their gender identity.”

The school district additionally barred cisgender girls who prefer masculine clothing from participating in school activities. One senior girl was stopped from walking across the graduation stage, moments before it was her turn, because she was wearing pants. The district prevented another senior girl’s portrait from appearing in the school yearbook because she wore a tuxedo.

Wednesday’s complaint is the civil rights group’s second attempt to challenge the school district on its discriminatory dress code. The ACLU sued the district last year for blocking a transgender senior girl, L.B., from wearing a dress at her graduation ceremony. A federal judge in Gulfport later upheld the school’s sex-specific dress code policy.

“If I wasn’t going to wear a dress, I wasn’t going to go,” L.B. said in testimony. “I was shocked, sickened and never expected it. I assumed I would be allowed to.”

The complaint to the Department of Education’s Office of Civil Rights comes just weeks after the Biden administration updated its final changes to Title IX to explicitly include the categories of gender identity and sexual orientation for the first time. The department made these changes after mounting pressure from LGBTQ+ and womens’ advocacy organizations and said last month that the changes were necessary to keep schools safe and welcoming to all students.

The Harrison County School District did not immediately respond to a request for comment.

The new guidance, which is slated to take effect Aug. 1, also reinstitutes protections for student survivors of sexual assault and harassment, which Donald Trump had rolled back during his presidency.

Trump has vowed to roll back protections once again for transgender students “on day one” ― and has hinted at a series of other executive orders he could make to jeopardize LGBTQ+ rights if he is elected president again in November.

The Title IX amendments now directly conflict with Mississippi’s transgender bathroom law, which prohibits a student from using facilities that don’t correspond with their sex assigned at birth. The law was signed Monday.

A number of other Republican-led states have enacted similar laws and restrictions on students using their chosen name and pronouns in the classroom.

Dozens of Republican officials have rejected the Biden administration’s update to the federal civil rights law and have sought various legal avenues to try to uphold anti-trans state policies. At the end of April, Texas Attorney General Ken Paxton sued the U.S. Department of Education, saying in a news release that the Biden administration was destroying protections for women in schools by “mandating compliance with radical gender ideology.”

Days later, Arkansas Gov. Sarah Huckabee Sanders signed an executive order to instruct schools to keep enforcing state laws barring transgender students from using bathrooms and joining sports teams that align with their gender identity. The Republican governor wrote that the Title IX rules are “plainly ridiculous” and will “lead to males unfairly competing in women’s sports.”

This week, four state attorneys general (in Kansas, Utah, Wyoming and Alaska) and the conservative organizations Moms for Liberty, Young America’s Foundation and Female Athletes United filed an additional lawsuit over the regulations. The suit is supported by the Alliance Defending Freedom, a conservative legal organization that helped overturn Roe. v. Wade two years ago and has been at the forefront of legal opposition to trans athletes in girls’ sports and youth access to gender-affirming care.