The aim of this study was to investigate the presence of NTM in the lymph nodes of opossums (D. virginiana) and to characterise the microscopic changes in affected tissue. Retropharyngeal and tracheobronchial lymph nodes were collected postmortem from 18 opossums in the state of Colima, Mexico in 2013. The lymph nodes were also cultured for mycobacterial organisms and processed for histopathological examination. Bacteriological cultures yielded 5/18X100 (28%) isolates of NTM, which were subsequently identified as M. terrae, M. szulgai, M. gastri and M. asiaticum. Microscopic examination of the affected nodes revealed a necrotic granulomatous lymphadenitis (3/60%) composed of histiocytes, epithelioid cells and giant cells with intralesional alcoholresistant acid bacteria. An association between the sex of the opossum and the presence of NTM was observed. To our knowledge, this is the first report of NTM isolation in opossums with granulomatous lymphadenitis in Mexico.