Exposure to stigma, violence, sex work, and substance use are associated with increased HIV risk, but relationships between these factors have not been fully elucidated among transgender women whose data are often aggregated with men who have sex with men and other sexual and gender minorities. Considering this gap, we aimed to identify a serologically confirmed HIV estimate for transgender women and examine the relationships between stigma, sex work, substance use, and HIV among a national sample of transgender women in Dominican Republic. We analyzed biomarkers and self-report data from the third wave of Dominican Republic's Encuesta de Vigilancia y Comportamiento con Vinculación Serológica, employing logistic and negative binomial regression to estimate models (n = 307). HIV rate was 35.8%. Nearly 75% of respondents engaged in sex work. Over 20% reported experiencing violence; 61.6% reported being stigmatized. Participation in sex work was associated with higher levels of stigma [incidence rate ratio (IRR): 1.70, p < 0.05]. Respondents who experienced violence had over three times higher odds of living with HIV relative to respondents who had not been victimized [odds ratio (OR): 3.15, p < 0.05]. Marijuana users were less likely to experience stigma compared with cocaine users (IRR: 1.72, p < 0.05), and a higher risk of alcohol dependency was associated with higher odds of experiencing violence (OR: 1.17, p < 0.001). Findings illustrate the importance of disaggregating data collected from transgender women compared with other sexual and gender minorities to ascertain subpopulation-specific estimates and indicate an urgent need to implement structural interventions and policies to protect transgender women's health and their human rights.