Bovine babesiosis is an important tick-borne disease caused by the parasites belonging to the genus Babesia, distributed worldwide and infecting a wide range of domesticated and wild cattle, occasionally man. The present study was conducted to determine the prevalence of B. bigemina infection in apparently healthy cattle from central plain zone of Punjab, India. Examination of Giemsa-stained peripheral thin blood smears revealed 2.45 % (5/204) animals to be positive for piroplasms of B. bigemina. However, genomic DNA isolated from these blood samples when subjected to primary PCR revealed a positivity of 7.35 % (15/204) as detected by the amplification of a 278-bp product in the agarose gel. PCR products obtained from the primary PCR of B. bigemina, when employed as template in nested PCR produced the amplicons of desired size (170 bp) was detected in 30.39 % (62/204) of the samples. It can thus be concluded that B. bigemina infection is prevalent in apparently healthy cattle population of this region and PCR assays can serve as a valuable tool for epidemiological studies in endemic areas.