BackgroundChina is a country with high burden of tuberculosis (TB), especially drug‐resistant TB (DR‐TB), which is still a serious health problem in Yunnan Province. Mycobacterium tuberculosis (MTB) is the pathogenic microorganism of TB. The epidemiological characteristics of MTB strains in local areas need to be described.MethodsA total of 430 clinical MTB isolates were collected from Yunnan Province and genotyped through the method of 24‐locus mycobacterial interspersed repetitive unit‐variable number tandem DNA repeats (MIRU‐VNTR).ResultsThe genotypes of the 24 loci showed abundantly genetic diversity, and allelic diversity index (h) of these loci varied from 0.012 to 0.817. Among the 430 strains, 30 clusters and 370 unique genotypes were identified. Beijing family was the predominant lineage (70.47%) in Yunnan MTB strains, and the other lineages contained T family (5.81%), MANU2 (0.70%), LAM (3.26%), CAS (0.23%), New‐1 (8.37%), and some unknown clades (11.16%). A total of 74 TB strains were identified as drug resistance through drug susceptibility testing (DST), including 38 multidrug‐resistant TB (MDR‐TB) and 36 single‐drug‐resistant TB (SDR‐TB). The frequency of MDR‐TB strains was significantly higher in Beijing family (10.89%) than that in non‐Beijing family (3.94%, P = 0.032).ConclusionsAlthough MTB strains showed high genetic diversity in Yunnan, China, the Beijing family was still the dominant strain. A high frequency of MDR‐TB strains was recorded in the Beijing family.