Canine babesiosis is an important tick-borne protozoal disease of dogs that poses major health problem worldwide. Farm dogs in rural areas are the companion animals, that not only watch the livestock herds but also guard the house of the owners. Each farmer keeps his companion dog to get all the services. In our study, a total of 450 blood samples of farm dogs from three different ecological zones (Southern, Central and Northern regions of the province; Punjab) of Pakistan, were collected to examine through microscopy and PCR. Examination of thin blood smears revealed an overall prevalence of 12.8% (58/450) of canine babesisal parasites. However, PCR analysis revealed 46.8% (211/450) and 7.3% (33/450) samples positive for B. gibsoni and B. vogeli, respectively. The amplicons of 671 bp and 590 bp were amplified for the detection of B. gibsoni and B. vogeli, respectively through PCR. The results of multivariate analysis showed that the occurrence of canine babesiosis is higher in the Central Punjab and younger age of the dogs, while breed and sex of the host were not significantly associated with the occurrence of the disease. Mixed infection of B. gibsoni and B. vogeli was observed only in 3 dogs each in district Kasur and Rawalpindi. Our study is the first report to observe the occurrence of canine babesiosis in rural dogs in Pakistan through PCR.