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Abstract Introduction Leptospirosis is a neglected emerging and zoonotic disease reported worldwide. This study sought to determine the molecular and serological prevalence of Leptospira spp. and the associated risk factors in slaughtered cattle from the Bahr El Ghazal region of South Sudan. Materials and methods Between January 16th and February 25th, 2023, blood and urine samples were collected from 402 cattle at the Lokoloko Municipal Slaughterhouse in Western Bahr El-Ghazal State. Serum samples were tested using the microscopic agglutination test (MAT), with a panel of 12 serovars (sv) from 12 serogroups (sg) and 4 species (spp) of Leptospira spp. These serovars had been previously identified in Sudan and the East African region. Simultaneously, 400 corresponding urine samples were screened using qualitative real-time polymerase chain reaction (PCR) to detect the shedding of Leptospira spp. in urine. To identify the associated risk factors, the age, sex, breed and body condition score of each sampled cattle was noted at the time of sampling and subsequently analysed using logistic regression models. Results Among the 402 serum samples screened, a substantial 81.8% (329/402, 95% CI 77.9–85.3) displayed seropositivity for Leptospira spp. with a MAT titre ≥ 100. The prevalence of urine shedding determined by PCR was 6% (23/400, 95% CI 3.8–8.4), while probable recent leptospirosis with a MAT ≥ 1:800 was observed in 33.1% (133/402, 95% CI 28.6–37.8) of the cattle. Multiple reactions were detected in 34.8% (140/402, 95% CI 30.6–39.5) serum samples. The seropositivity was against L. borgpetersenii sg. Tarassovi (78.6%; 316/402, 95% CI 74.4–82.3), followed by L. borgpetersenii sg. Ballum at 20.4% (82/402, 95% CI, 16.7–24.4%), L. kirschneri sg. Autumnalis At 8.7% (35/402, 95% CI 5.7–11.7), L. interrogans sg. of Pomona at 7.0% (28/402, 95% CI 4.5–9.5), and L. interrogans sg. Hebdomadis was 5.0% (20/402, 95% CI 2.8–7.2). Several risk factors are associated with seropositivity. Older animals (≥ 2 years) had 2.0 times greater odds (95% CI 1.14–3.5) of being seropositive than younger animals (