The present study was conducted to identify the bovine Mycoplasmas/Acholeplasmas on clinical examination and PCR based laboratory confirmation from 1590 buffaloes and 470 lung samples collected from Abattoir Karachi during 2014 to 2016. Clinical field survey along with macroscopic examination revealed that total 467 (13.48%) buffaloes had signs of respiratory distress including sticky nasal discharge, sunken eyes, dyspnea and fever. Out of 470 lung samples collected from slaughter house183 (16.76%) were found culture positive for Mycoplasma species. All the culture positive samples were further evaluated by PCR using universal mycoplasma primers. PCR revealed that 201 (5.80%) nasal discharge from buffaloes and 142 (13.00%) lung tissues collected from abattoir were positive for mycoplamas infection. The PCR for Acholeplasma laidlawii was also performed using specie specific primers and the amplified product was sequenced for re-validate the specie. This revealed that 92 (2.66%) nasal discharge samples were positive; and 44 (4.03%) lung samples were positive for Acholeplasma laidlawii. It is concluded that the frequently isolation and molecular identification of Acholeplasma laidlawii from nasal discharge and lung tissue samples are very significant and this study also indicated alarming months for dairy farmers for precautionary measures.