Background Autochthonous cutaneous and visceral leishmaniasis caused by Leishmania martiniquensis and Leishmania siamensis have been considered an emerging infectious disease in Thailand. The disease burden is significantly underestimated, especially the prevalence of Leishmania infection among HIV-positive patients.Methods A cross-sectional study was conducted to determine the prevalence and risk factors associated with Leishmania infection among patients with HIV/AIDS living in Trang Province, southern Thailand between 2015 and 2016. Antibodies against Leishmania infection were assayed using the direct agglutination test (DAT). DNA of Leishmania was detected by ITS1-PCR using the buffy coat. Species of Leishmania were also identified.Results Of 724 participants, the prevalence of Leishmania infection was 25.1% (182/724) using either DAT and/or PCR assays. Seroprevalence of Leishmania infection was 18.5% (134/724), while Leishmania DNA detected by PCR method was 8.4% (61/724). Of these, 24.9% (180/724) were asymptomatic, whereas 0.3% (2/724) were symptomatic VL and VL/CL. At least five species were identified, that is, L. siamensis, L. martiniquensis, L. donovani complex, L. lainsoni, and L. major. Multivariate analysis showed that CD4+ levels