Ethiopia has a high burden of visceral leishmaniasis. Recently, there was a significant increase in cases in the South Omo Zone. This study aims to assess the prevalence of Leishmania donovani infection and its associated factors. A household-based cross-sectional study was carried out in January 2023 in the South Omo Zone in Ethiopia. Dried blood spot samples were collected from 382 randomly selected study participants. Direct agglutination test (DAT) and kinetoplast DNA real-time PCR tests were performed to detect L. donovani infection. Participants' sociodemographic, clinical and risk factors for L. donovani infection data were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyze the data. Febrile cases were checked for malaria with a multiplex PCR assay. Overall prevalence of L. donovani infection among the sampled population was 32.5% (n=124), of which 41.1% (n=51) was detected by PCR, 33.9% (n=42) by DAT and 25.0% (n=31) by both tests. The majority of the positives were from the Logira (28.2%; n=35) and Dilbayne (29.0%; n=36) villages. Participants residing in Logira (adjusted OR [AOR]: 5.80; 95% CI 1.85 to 18.15) and Dilbayne (AOR: 3.38; 95% CI 1.15 to 9.96) villages and owning cows (AOR: 2.31; 95% CI 1.03 to 5.15) showed an association with Leishmania infection. Plasmodium falciparum was detected in 3.4% (n=2) of 59 febrile participants. The prevalence of L. donovani infection in the South Omo Zone is high. Further research on the role of cows in the transmission cycle is needed to design the best strategy to control Leishmania infection in the South Omo Zone. Such interventions should focus on the Logira and Dilbayne villages, where most of the infections were identified.