Contagious bovine pleuropneumonia (CBPP) is a highly contagious disease of cattle caused by Mycoplasma mycoides subsp. mycoides. It is characterized by anorexia, fever, dyspnea, polypnea, cough, and nasal discharges. Gross lesions in the lung such as marbling, sequestra, thickening of interlobular septa, and consolidation are evident. Serological tests including complement fixation test and competitive enzyme-linked immunosorbent assay and molecular tests such as polymerase chain reactions are used for diagnostic purposes. In this study, lung samples of suspected large ruminants (cattle n=560, buffalo n=293) were collected from abattoirs of three districts of Punjab namely Lahore, Kasur and Jhang. PCR was performed with specific primers, targeting the 16S ribosomal RNA gene to detect the positive cases. The results indicated that 49 samples (8.75%) of cattle were positive, with maximum prevalence was observed in Jhang with 16 positive samples (10.06%), but CBPP was not detected in any buffalo sample. High prevalence of disease was seen in cattle of more than seven years of age, in female cattle, and in cross-bred cattle. Age and gender were found significantly associated (P<0.05) with the prevalence of the disease. Gene sequencing of identified 5 isolates of Mycoplasma mycoides subsp. mycoides had more than 99% similarities with the strains isolated from China, Italy, Australia and Tanzania and were categorized into a monophyletic group but strain isolated from Portugal had more than 55% variable regions, hence clustered separately. This study confirms the presence of contagious bovine pleuropneumonia in the country which can be a threat to the livestock export market and warrants the implementation of control measures to mitigate the economic losses associated with the disease.