Transgender (trans) women experience unique barriers in accessing preventative health services such as HIV preexposure prophylaxis (PrEP). These barriers may be exacerbated by past real or anticipated mistreatment in health care settings, but little is known about the relationship between medical mistrust and poor PrEP uptake and knowledge. Using a multistep approach, this study used a novel survey instrument administered to a pilot sample of 78 trans women. Item responses on a 0-10 scale were subjected to a TwoStep cluster analysis to explore how perceptions of PrEP and experiences with health care vary among trans women. Two distinct clusters (C1,C2) were defined on the basis of race (C1: 82% White, C2: 69% Black) and highest level of education completed (C1: 53% college or above, C2: 42% high school diploma or GED). Analyses suggest that varying levels of medical mistrust exist between clusters. Higher mean scores on medical mistrust items were reported in C1. A similar relationship was found on attitudes toward PrEP. Differences in intention to use PrEP and differences in past PrEP use were not significant; however, C2 members were more likely to have heard of PrEP from a doctor. Results suggest that levels of medical mistrust and PrEP perceptions vary among distinct subpopulations in this community, which may affect willingness to use PrEP. Interventions aimed at addressing unique perceptions in subpopulations could move trans women from intention to PrEP use.