This study assessed disparities in pre-exposure prophylaxis (PrEP) use among transgender and gender expansive youth and young adults (N = 477) between 15 and 24 years old in the CARES (ATN 149) and TechStep (ATN 160) study protocols within the National Adolescent Medicine Trials Network for HIV/AIDS Interventions (ATN). Structural equation modeling was used to test mediation pathways between gender identity and PrEP uptake among the full sample and stratified by sex assigned at birth. Lifetime PrEP uptake was higher among those assigned male at birth (26%) versus assigned female at birth (9%), explained by greater structural and behavioral risks and perceived need for PrEP, especially among trans women. Among those assigned female at birth, PrEP uptake was higher among trans men (12%) than nonbinary participants (6%). Our findings characterize key structural and behavioral drivers of PrEP use and highlight the need to reduce barriers to healthcare for trans youth, particularly in the South.