Surra is an infectious disease caused by Trypanosoma evansi (T. evansi), which is spread in developing countries. This study aimed to estimate the prevalence and genetic characterization of T. evansi in cattle in Makkah, Saudi Arabia. Blood samples from 150 cattle were collected and examined by a blood smear and PCR targeting the RoTat 1.2VSG gene. The findings revealed that the prevalence of T. evansi varied depending on the detection test, ranging from 20.7% to 25.3% by blood smear examination and PCR. RoTat 1.2VSG sequences analysis of two T. evansi isolates revealed little difference compared to similar sequences in the database. The phylogenetic tree revealed that local T. evansi isolates in this study were clustered with other T. evansi sequences from Egypt, India, Nigeria, Kenya, and Pakistan. The sequences in the study and the Egyptian sequence (MG674485, camel strain) were grouped into a single clade with little genetic variation. In conclusion, the results of this study urge for the introduction and adoption of appropriate control strategies to reduce the effect of T. evansi infection on cattle production in Saudi Arabia.