Background: There are seven human-adaptation lineages of Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) dissemination is strongly influenced by human movements and host genetics. The detailed lineage distribution evolution of Mtb in Zhejiang Province is unknown. We aim to determine how different sub-lineages are transmitted and distributed within China and Zhejiang Province.</p><p>Methods: We analysed whole-genome sequencing data for a worldwide collection of 1154 isolates and a provincial collection of 1296 isolates, constructed the best-scoring maximum likelihood phylogenetic tree. Bayesian evolutionary analysis was used to calculate the latest common ancestor of lineages 2 and 4. The antigenic diversity of human T cell epitopes was evaluated by calculating the pairwise dN/dS ratios.</p><p>Results: Of the Zhejiang isolates, 964 (74.38%) belonged to lineage 2 and 332 (25.62%) belonged to lineage 4. The distributions of the sub-lineages varied across the geographic regions of Zhejiang Province. L2.2 is the most ancient sub-lineage in Zhejiang, first appearing approximately 6897 years ago (95% highest posterior density interval (HDI): 6513-7298). L4.4 is the most modern sub-lineage, first appearing approximately 2217 years ago (95% HDI: 1864-2581). The dN/dS ratios showed that the epitope and non-epitope regions of lineage 2 strains were significantly (P<0.001) more conserved than those of lineage 4.</p><p>Conclusions: An increase in the frequency of lineage 4 may reflect its successful transmission over the last 20 years. The recent common ancestors of the sub-lineages and their transmission routes are relevant to the entry of humans into China and Zhejiang Province. Diversity in T cell epitopes may prevent Mycobacterium tuberculosis from being recognized by the immune system.