Leishmaniasis poses significant public health challenges in endemic regions. Understanding the prevalence of asymptomatic Leishmania infection and identifying risk factors among blood donors is crucial. This study addressed a knowledge gap by evaluating the prevalence of asymptomatic Leishmania infection and pinpointing associated risk factors among blood donors in an endemic area in Thailand and aimed to enhance blood donation safety protocols and reduce the risk of transfusion-transmitted Leishmania infection. A cross-sectional study and a longitudinal follow-up were conducted among 500 blood donors in Trang Province, southern Thailand. A serological test was performed using the direct agglutination test (DAT), and DNA detection was performed using nested polymerase chain reaction (nPCR) to screen for Leishmania infection. Potential risk factors associated with the infection were also assessed. The study identified a 19.0% prevalence of asymptomatic Leishmania infection among blood donors, with nPCR proving more effective in detecting infections (13.0%) than DAT (6.4%). Notably, Leishmania martiniquensis was the predominant species identified, highlighting the local epidemiological profile of Leishmania infection. Furthermore, using multivariate analysis, living in stilt houses was independently associated with Leishmania infection (adjusted odds ratio = 1.85; 95% CI = 1.04-3.28; P = 0.035). A high prevalence of asymptomatic Leishmania infection among blood donors underscores the need for integrating comprehensive Leishmania screening protocols into blood donation processes, particularly in endemic regions. It advocates for using molecular diagnostics to enhance detection accuracy. Furthermore, living in stilt houses as a risk factor emphasizes the importance of environmental management in leishmaniasis control efforts.