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Objective To understand the molecular biological characteristics of multidrug-resistant Mycobacterium tuberculosis in Urumqi by the analysis of gene mutation and clustering of multidrug-resistant Mycobacterium tuberculosis in Urumqi, to provid theoretical basis for the prevention and control of multidrug-resistant tuberculosis in the future. Methods The tuberculosis patients who were diagnosed, registered, cultured positive Mycobacterium tuberculosis and over 16 years old in Urumqi were studied. Geno Type MTBDR plus Assay and MIRU-VNTR were used to analysis the gene mutation and clustering of multidrug-resistant mycobacterium tuberculosis in Urumqi. Result Mutation gene included Isoniazid resistance gene katG 315; Rifampicin resistance gene rpoB 526, 531; Ethambutol resistance gene embB 206; Streptomycin resistance gene rpsL 43 and RRs 1401; Ofloxacin resistance gene gyrA 74, 90, 91 and 94. It was shown that the Beijing genotype was predominant (49.530%) among these319strains. There were 14 strains multi-drug resistant strains, it was predominant (73.684%) of multi-drug resistant strains. Multi-drug resistant prevalence of Beijing strains was 8.861%. The clustering rate of MDR-TB was 5.263% in Urumqi. Conclusion Molecular biological characteristics of multidrug-resistant Mycobacterium tuberculosis have some particularities in Urumqi. Therefore, modern molecular epidemiological techniques such as molecular biology and DNA fingerprinting can be used to improve the speed of diagnosis and to carry out case tracking and source investigation.