Background In Thailand, HIV transmission is well characterized in large urban centers such as Bangkok and Chiang Mai but less so outside of these areas. We assessed HIV incidence and associated risk factors in two other locations. Methods Participants assigned male sex at birth were enrolled in Nakhon Ratchasima and Ratchaburi. HIV and syphilis testing and sociobehavioral questionnaires occurred over 18 months. HIV incidence rates and 95% confidence intervals (CIs) were estimated using a Poisson distribution. Cox proportional hazards models were used to estimate unadjusted and adjusted hazard ratios (aHRs) and 95% CIs for associations between potential risk factors and HIV seroconversion. Results A total of 1003 participants were enrolled. Overall HIV incidence was 1.56 per 100 person-years (95% CI:1.02–2.44) and similar at both sites. In the fully adjusted model, receptive anal sex in the past 6 months was associated with seroconversion (aHR:3.05, 95% CI:1.00-9.32), while sex with a sex worker in the past six months was associated with reduced risk of seroconversion (aHR:0.11, 95% CI:0.01–0.80). In the parsimoniously adjusted model, receptive anal sex (aHR:3.35, 95% CI:1.30–8.63) and STI diagnosis in the past six months (aHR:3.39, 95% CI:1.11–10.29) were associated with seroconversion, while sex with a sex worker in the past six months was associated with reduced risk of seroconversion (aHR:0.12, 95% CI:0.02–0.70). Conclusions Recent receptive anal sex practices were associated with HIV acquisition in these populations, highlighting the continued need for interventions encouraging safer anal sex practices to reduce HIV incidence.