The present study aimed to estimate the molecular prevalence of Theileria annulata and Trypanosoma evansi infection in cattle in Northern Tunisia. A total number of 96 cattle from five farms were evaluated. T. annulata and T. evansi prevalences were 61% [56/66] and 10% [7/13], respectively, at a confidence interval (CI) of 95%, while co‐infection was present in 6% [4/8] of the tested animals at a CI of 95%. There was a significant correlation between age and the prevalence of T. annulata infection, whereas, there was no significant association shown with the age of cattle and T. evansi infection. Sequence and phylogenetic analyses showed that the T. annulata Tams1 gene and T. evansi
ITS1 rDNA gene were highly conserved with 97.1–100% and 98.3–100% sequence identity, respectively.