Background The syphilis epidemic continues to cause substantial morbidity worldwide and is worsening despite ongoing control efforts. Syphilis remains an important public health problem among 3 key populations: men who have sex with men (MSM), transgender women, and female sex workers. Methods We conducted a retrospective chart review of patients that received rapid point-of-care treponemal antibody tests from January 2019 to July 2019 in 4 sexually transmitted infection (STI) clinics in Lima, Peru. We assessed patient medical records for human immunodeficiency virus (HIV) infection, history of STIs, as well as sociodemographic and behavioral characteristics. Cross-sectional descriptive analyses were used to determine factors associated with treponemal positivity. Results We included 401 patient records in our analyses: 252 MSM, 31 transgender women, and 118 female sex workers. The overall median age of patients was 29.0 years (interquartile range, 24.0-36.0 years). Positivity on the treponemal test was 28.9% (95% confidence interval [CI], 24.3%-33.3%) overall, 37.7% (95% CI, 31.7%-44.0%) for MSM, 54.8% (95% CI, 36.0%-72.7%) for transgender women, and 3.4% (95% CI, 0.9%-8.5%) for female sex workers. In the bivariate analysis, treponemal positivity was also associated with receptive anal sex in the last 6 months in MSM (P < 0.01). Additionally, treponemal positivity increased with age (P = 0.0212) and varied by socioeconomic status (P < 0.01). Multivariate Least Absolute Shrinkage and Selection Operator logistic regression showed that treponemal positivity was highly associated with HIV coinfection (adjusted odds ratio, 5.42) and previous STI other than HIV or syphilis (adjusted odds ratio, 1.54). Conclusions A review of the medical records of members of 3 key populations who had recently received a rapid point-of-care treponemal test in Lima, Peru, revealed that lifetime prevalence of syphilis was high among MSM and transgender women, but low among female sex workers. Those results may indicate a need for more frequent, regular testing among MSM and transgender women - possibly in conjunction with HIV testing, and appropriate treatment of those shown to be positive.