OBJECTIVE To develop a new DNA chip for rapid detection of rpoB,katG and inhA gene mutation in rifampin and/or isoniazid-resistant Mycobacterium tuberculosis isolated from clinical laboratorial specimen.METHODS We designed 16 oligonucleotide probes specific for detection of the mutant sequences in genes rpoB,katG and inhA.RESULTS Mutations were found in 26 strains(100%) of 26 randomly selected rifampin-resistant M.tuberculosis and 24 strains(80%) of 30 randomly selected isoniazid-resistant M.tuberculosis by DNA chip.CONCLUSIONS DNA chip technology has high sensitivity and specificity in detection of rifampin-resistant M.tuberculosis and may be applied in clinical diagnosis.