The aim of this study was to characterize the genotypes of Mycobacterium tuberculosis (MT) in isolates obtained from Henan, China, and to study the distribution features of Beijing strains in Henan. A total of 443 MT strains isolated in Henan Province were tested for susceptibility to isonicotinylhydrazide (INH), rifampicin (RFP), ethambutol (EMB) and streptomycin (SM), and genotyped by spoligotyping. The clustering of genotypes revealed 4 gene clusters (Beijing and Beijing-like, T, Manu, and S and LAM3) and 24 genotypes. In total, 387 (87.4%) of the strains were Beijing strains. The frequency of multidrug-resistant (MDR) strains was significantly higher in the Beijing and Beijing-like strains than in the other strains (χ(2)=4.6564, P<0.05). However, the percentages of drug resistance and sensitivity in the Beijing strains were almost the same as those in the non-Beijing strains. The proportion of Beijing strains in the ≤60-year-old TB patients was significantly higher than in the >60-year-old TB patients (χ(2)=32.053, P<0.001). The distribution of Beijing strains deceased gradually from the east to the west in Henan Province (P<0.0001). The data demonstrate that the Beijing genotype is a major type in the area and may be related to enhanced transmissibility. The transmission of Beijing family strains has increased in Henan and its incidence is highest in the east of Henan. The MDR strains were significant in the spread of MT.