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Abstract Babesia spp. are tick-transmitted apicomplexan parasites that infect erythrocytes of a wide range of vertebrates including domestic animals, and can be associated with a considerable economic loss due to introduction of prophylactic measures, decreased production and rates of morbidity and mortality. An efficient detection assay needs to be set up for accurate diagnosis and prevention of disease. The present work is aimed at verifying the occurrence of Babesia bigemina infection in water buffaloes in the southeastern region of Pakistan by using both traditional blood smear and molecular techniques. A total of 100 blood samples were collected from tick-free and tick-infested animals (50 animals each group). The overall prevalence was recorded as 20% and 17% by using thin blood smear and PCR methods, respectively. Moreover, the analysis of infection in tick-infested and tick-free animals showed the infection rates of 36% and 4% by using blood smear method, and 30% and 4% with that of PCR method, respectively. Despite the higher prevalence rate recorded by blood smear method, which may be the result of non-specific identification of Babesia species, these results indicate that the PCR assay used in this study provides a useful tool for accurate diagnosis of the Babesia bigemina infection in bovines. Key words: Babesia bigemina, diagnosis, PCR, prevalence, blood smear technique