Yunnan is a province located in southwestern China. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis (MTB) strains circulating in Yunnan Province. We used spoligotyping and a 12-locus high-resolution VNTR set to identify a total of 271 MTB isolates collected from six prefectures' Yunnan. All the 271 patients were classified as 11 different ethnic groups, including 133 Han patients (49.1%) and 138 minority patients (50.9%). Spoligotyping analyses revealed that the largest two spoligotypes were SIT1 (Beijing family, n = 136) and SIT53 (T family, n = 35). Statistical analysis indicated that the proportion of Beijing genotype in Qujing was significantly higher than that in the ethnic enclaves (P ≤ 0.01). Compared with the percentage of Beijing-family isolates from patients under 25 years of age (72.7%), those from patients aged 45~64 years (44.6%, P < 0.01) and older than 64 years (48.1%) (P = 0.04) were significantly lower. Beijing genotype strains (23.8%, 36/151) showed higher clustering rate than non-Beijing genotype strains (16/120, 13.3%, P = 0.029). In conclusion, our data demonstrated that the Beijing genotype was the predominant genotype in Yunnan Province. The distribution of Beijing genotype strains showed geographic diversity. In addition, Beijing genotype was associated with recent transmission rather than drug resistance.