Mycobacterium bovis strains isolated from tuberculosis (TB) lesions from 1138 cattle slaughtered at Kombolcha abattoir in north eastern Ethiopia were characterised. Detailed postmortem examination, bacteriological culturing, regions of difference PCR and spoligotyping were used. At least one TB lesion was observed in 57 (5 per cent) of the cattle, of which 27 (47 per cent) yielded mycobacteria isolates. Of the 27 isolates, 25 were identified as M bovis and two as Mycobacterium tuberculosis. The M bovis isolates were grouped into six clusters of strains, and the M tuberculosis isolates were typified to one strain only with the reference SIT262. Three clusters of previously unreported M bovis strains were identified and reported to the Mycobacterium bovis spoligotype database. They were designated the reference numbers SB1490, SB1491 and SB1492.