The prevalence of Trypanosoma evansi was investigated in 1,250 Nili-Ravi buffaloes of mixed age and sex by polymerase chain reaction (PCR) for the first time in Pakistan. DNA of the trypanosomes was isolated with TRIREAGENT®. The assay was employed using primers ESAG 6/7, specific for a 237-bp fragment from T. evansi genomic DNA. The samples were screened for the presence of T. evansi also by stained thin smear. Forty-four (3.5%) samples were positive by microscopy, while 97 (7.7%) samples were identified by PCR, indicating the high sensitivity of PCR for surveying the disease in epidemiological studies.