To detect rifampicin-resistant mutations in Mycobacterium kansasii (M. kansasii).We examined the M. kansasii isolates from sputum of patients at National Hospital Organization Kinki-chuo Chest Medical Center from January 1, 2001 to November 30, 2005 using drug-susceptibility testing, and analyzed 69-bp fragment of rpoB gene in rifampicin-resistant strains.Three strains from 314 isolates were determined as rifampicin resistant using drug-susceptibility testing. Those strains showed a rise in minimum inhibitory concentration (MIC), and had the mutations in rpoB gene. These point mutations in codons 513 and 516 were common mutations found in rifampicin-resistant clinical isolates of M. tuberculosis.We verified the association between rpoB gene mutations and rifampicin resistance in M. kansasii.