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Canine babesiosis is a serious threat to dogs&#8217; health worldwide, caused by the intra-erythrocytic <i>Babesia</i> species. The present study was carried out in pet dogs presented at three clinics of Lahore and one of Narowal in Punjab, Pakistan. Two hundred blood samples (50 from each clinic) were collected and screened by microscopy for <i>Babesia</i> spp. Out of 200 samples, 84 (42%) were found to be positive for babesiosis. The highest number of positive cases (50%) was recorded in dogs at Narowal clinic. Non-significant variation (<i>p</i> &gt; 0.05) was observed in the prevalence of babesiosis in dogs in relation to sex and age. Positive samples were further confirmed by a polymerase chain reaction using 18S-rRNA genus-specific and species-specific primers. Amplicons were further analyzed by nucleotide sequencing for genetic diversity. <i>Babesia canis</i> and <i>gibsoni</i> were confirmed by genome sequencing in all diseased dogs. These isolates closely resembled each other, but differed from previous reported strains. In conclusion, pet dogs suffering from babesiosis were infected with <i>B. canis</i> and <i>gibsoni</i>, while in other countries, other <i>Babesia</i> species are also prevalent.