The 15th natural plague focus in China, the Junggar Basin plague focus, is located near an important communication route connecting China and Central Asia and was discovered after 2005. To characterize the phenotypic and genetic diversity of the Yersinia pestis population in this newly established focus, we collected 25 Y. pestis strains from six counties across Junggar Basin in 2005-2006, and determined their biochemical features and genotypes based on multiple-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindromic repeats analysis. We inferred the phylogenetic positions and possible sources of the Junggar strains by comparing their genotypes with the genetic diversity for known representative Y. pestis strains. Our results indicate that the major genotype of Junggar strains belongs to 2.MED1, a lineage of biovar Medievalis with identical biochemical characters and high virulence in mice. Although share a similar ecology, the 2.MED1 in Junggar Basin are not descended from known strains in the neighboring Central Asian Desert plague foci. Therefore, the emergence of the Junggar Basin plague focus is not attributable to the recent clonal spread of Y. pestis from Central Asia. We also identified two distinct minor genotypes in Junggar Basin, one of which clusters genetically with the 0.ANT1 strains of the Tianshan Mountain natural plague focus and another belongs to a 1.IN lineage not previously reported. Our study clarifies the phenotypic and genetic characters of Junggar Y. pestis strains. These findings extend our knowledge of the population diversity of Y. pestis and will facilitate future plague surveillance and prevention in Junggar Basin and adjacent regions.