Objective:To determine the presence of Babesia bovis (B. bovis) in large ruminants in south Punjab and its effect on hematological and serum biochemical profile of host animals. Methods:Blood samples were collected from 144 large ruminants, including 105 cattle and 39 buffaloes, from six districts in south Punjab including Multan, Layyah, Muzaffar Garh, Bhakar, Bahawalnagar and Vehari. Data on the characteristics of animals and herds were collected through questionnaires. Different blood (hemoglobin, glucose) and serum (ALT, AST, LDH, cholesterol) parameters of calves and cattle were measured and compared between parasite positive and negative samples to demonstrate the effect of B. bovis on the blood and serological profile of infected animals. Results:27 out of 144 animals, from 5 out of 6 sampling districts, produced the 541-bp fragment specific for B. bovis. Age of animals (P=0.02), presence of ticks on animals (P=0.04) and presence of ticks on dogs associated with herds (P=0.5) were among the major risk factors involved in the spread of bovine babesiosis in the study area. ALT concentrations were the only serum biochemical values that significantly varied between parasite positive and negative cattle. Conclusions:This study has reported for the first time the presence of B. bovis in large ruminant and the results can lead to the prevention of babesiosis in the region to increase the livestock output.