Plague is a natural focus-based disease, and for better understanding of this disease it is crucial to determine the molecular mechanisms of its pathogen, Yersinia pestis, for adapting to different foci. Gene inactivation, loss and acquisition are the main mechanisms that contribute to a pathogen's fitness. Determination of the whole-genome sequences of three Y. pestis strains, CO92, KIM and 91001, provided a good opportunity to probe into its genome in minute detail. Many genetic variations were found between the three strains. The present work focused on adaptive microevolutionary analysis of Y. pestis from different natural plague foci in China based on pseudogene profiles. Twenty-four mutations that led to inactivation in the corresponding genes were analysed, and a PCR-based screening method was employed to investigate the distribution of these mutations among Y. pestis isolates from different foci and also among seven strains of Yersinia pseudotuberculosis. It was found that Y. pestis isolates from the same focus had identical mutation profiles, and 260 isolates of Y. pestis were divided into eight genotypes, while Y. pseudotuberculosis harboured wild-type alleles for all the mutations. The isolates of three known biovars were grouped into distinct branches in the phylogenetic tree, which supports the proposition that biovars mediaevalis and orientalis directly arose from biovar antiqua individually. The constructed phylogenetic tree suggests that the isolates from focus B should be the oldest lineage of Y. pestis in China except for isolates from foci L and M, which might be a special lineage of Y. pestis and originated differently to the others.