Babesiosis is endemic in Pakistan and is one of the most important bovine diseases that cause huge economic losses and high mortality in young animals. A hematobiochemical study was conducted to unveil the difference between diseased and healthy animals in specific districts i.e., Faisalabad, Toba Tek Singh and Jhang of Punjab, Pakistan. The total number of blood samples collected were 518 which include samples of 158 buffaloes and 360 cattle. The collected samples are then analyzed with the help of PCR (Polymerase Chain Reaction) targeting apocytochrome b-genes (CYTb) followed by hematobiochemical analysis. The analysis of data was done by the Chi-square test. The PCR tests conducted in summer suggests that 53 out of 180 (29.4%) cows and 19 out of 79 (24.05%) buffaloes are prone to babesiosis. On the other hand, in winter results showed that 12.7 (23/180), 13.92 % (11/79) samples positive for Babesia genus through c-PCR. The positive samples were further investigated for hematological and biochemical analysis. The results revealed that, the mean value of hematological parameters like RBCs, Hb, PCV, MCV and MCHC was significantly (P< 0.05) decreased in infected animals (cows and buffaloes) as compared to the non-infected ones. While the biochemical parameters like ALT, AST, Cholesterol and LDH were significantly (P< 0.05) increased in infected animals as compared to healthy animals. These findings are the novel molecular and hematobiochemical evidence of B. bovis in dairy herds of Punjab province, Pakistan.