Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray.Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains.Target DNA of M.tuberculosis was amplified by PCR,hybridized and scanned.Direct sequencing was performed to verify the results of oligonuclcotide microarray.Results of the 102 rifampin-resistant strains 98 (96.1%) had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M.tuberculosis isolates.