Abstract This is a cross-sectional molecular epidemiological study on equine piroplasmosis (EP) affecting horses and donkeys in the Sudan. The study evaluated 499 samples from geographically distinct regions in eastern, central and western parts of the country. PCR amplification of the 18S rRNA gene of both Thelieria equi and Babesia caballi was carried out. Horses from all sampled areas were found positive to T. equi DNA but no B. caballi was detected. Absence of B. caballi infection was confirmed by another PCR targeting the B. caballi 48-kDa merozoite antigen. The overall prevalence was found to be 35.95%. The highest prevalence was detected in Showak 13 (81.3%) and the lowest was in Shearia locality in South Darfur 1 (5.6%). In another experiment, capillary electrophoresis was used to detect and differentiate between T. equi and B. caballi using one set of primers designed to amplify the 18S rRNA gene in a single PCR. Capillary electrophoresis method was found to be powerful in detecting mixed infections in artificially mixed controls samples. The data obtained in this study would contribute to the development of a national control strategy of EP in the Sudan.