Abstract: Objective To explore the genetic profile of tuberculosis from Henan. Methods 668 MTB isolates from various areas during 2015 were genotyped with spoligotyping and 26-locus mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) (classical 24-locus MIRU-VNTR and 2 other loci). The association between tuberculosis spoligotype signatures and drug-resistant profiles was analyzed. Results Spoligotyping data revealed 668 isolates were grouped into 10 different clusters. Beijing family was the most predominant genotype in Henan. There was no correlation between Beijing family and gender, age at diagnosis and treatment history, however, Beijing family had a higher proportion of resistance to all four first-line drug and multidrug-resistant phenotypes. 668 isolates were divided into 567 different types by 26-locus MIRU-VNTR analysis. For these samples, 15 of 26 MIRU-VNTR loci had highly or moderately discriminatory power according to the Hunter-Gaston discriminatory index. A combination of the 10 most polymorphic loci acquired similar discriminatory power to that of 26-locus set. Conclusions Beijing genotype is the most prevalent family. 10-locus MIRU-VNTR in combination with spoligotyping can efficiently investigate evolutionary genetics of MTB in Henan Province.