Latino men who have sex with men (LMSM) and Latina transgender women (LTGW) often lack access to HIV prevention information and strategies such as pre-exposure prophylaxis (PrEP). We explored knowledge gaps and culturally sensitive messaging about PrEP among HIV-negative LMSM and LTGW in Los Angeles. We recruited participants from a Latinx LGBT community-based organization. We conducted nine focus groups (n = 91 participants) with 52 LMSM and 39 LTGW. We used a rapid assessment process to create narrative reports that we analyzed using thematic analysis. Key quotes were transcribed verbatim; they were reviewed by the team, then uploaded to Dedoose to identify themes across sites and between groups. Three themes emerged for both LMSM and LTGW: knowledge gaps regarding PrEP remain; people who have knowledge about PrEP often served as its champions; highlighting positive aspects of culture could help improve PreP’s uptake and sustained use. Only LMSM worried that PrEP could impact condom use. Some issues were more pronounced among LTGW (e.g., more limited access to PrEP); others were unique to LTGW (e.g., worry about drug-hormones interactions). Collaborative research, programs, and policies, informed by LMSM and LTGW themselves, are needed to narrow existing knowledge gaps and promote PrEP uptake and sustained utilization.