BACKGROUND: Investigation of the genetic diversity of Mycobacterium tuberculosis in China has shown that Beijing genotype strains play a dominant role in the tuberculosis (TB) epidemic. In order to examine the strain diversity in the whole country, and to study the evolutionary development of Beijing strains, we sought to genotype a large collection of isolates using different methods. METHODOLOGY/PRINCIPAL FINDINGS: We applied a 15-loci VNTR typing analysis on 1,586 isolates from the Beijing municipality and 12 Chinese provinces or autonomous regions. The data was compared to that of 900 isolates from various other worldwide geographic regions outside of China. A total of 1,162/1,586 (73.2%) of the isolates, distributed into 472 VNTR types, were found to belong to the Beijing genotype family and this represented 56 to 94% of the isolates in each of the localizations. VNTR typing revealed that the majority of the non-Beijing isolates fall into two genotype families, which represented 17% of the total number of isolates, and seem largely restricted to China. A small number of East African Indian genotype strains was also observed in this collection. Ancient Beijing strains with an intact region of difference (RD) 181, as well as strains presumably resembling ancestors of the whole Beijing genotype family, were mainly found in the Guangxi autonomous region. CONCLUSIONS/SIGNIFICANCE: This is the largest M. tuberculosis VNTR-based genotyping study performed in China to date. The high percentage of Beijing isolates in the whole country and the presence in the South of strains representing early branching points may be an indication that the Beijing lineage originated from China, probably in the Guangxi region. Two modern lineages are shown here to represent the majority of non-Beijing Chinese isolates. The observed geographic distribution of the different lineages within China suggests that natural frontiers are major factors in their diffusion.