Mycoplasma bovis (M. bovis) is one of the important pathogens of cattle, which have been detected from pneumonic lungs of dairy cattle especially calves. M. bovis is main reason for morbidity and mortality in dairy calves because it is overlooked due to its late detection. It is considered that some typical clinical ailments, postmortem and microscopic lesions are useful for the diagnosis of disease but molecular confirmation through PCR is more reliable technique. The present study was conducted for detection and phylogenetic analysis of M. bovis induced mortality in bovine calves. A total of 193 lung and pleural fluid samples (bovine calves) were collected from slaughterhouses and private farms in Bahawalpur region. The DNA was extracted from samples by using DNA extraction kit. The samples were analyzed by PCR using M. bovis-specific primers having a length of 270bp. The PCR results showed that M. bovis was detected in 16(8.29%) isolates, which included 14(7.90%) lung tissues and 2(12.50%) pleural fluid samples. The season-wise positive samples were 12.12% in winter, 10.34% in spring and 2.89% in summer. The sequences were submitted to NCBI under accession number allotted as PP738878-PP738885. The BLAST was performed for sequence alignment by windows 7.1 software MEGA Muscle 37. The phylogenetic analysis showed the maximum and minimum identity of 97-100% among these geographical obtained strains from local isolates. The reference strain PP738878 had 100% identity with Swiss strain AF003959.1 of M. bovis and other strains showed a minimum identity of 99% with Poland, Switzerland and Egypt. This is the first study of the molecular detection and phylogenetic analysis of M. bovis in bovine calves in Pakistan.