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Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoB gene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.