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Background  Variable number of tandem repeats (VNTRs) that are widely distributed in the genome of  Yersinia pestis  proved to be useful markers for the genotyping and source-tracing of this notorious pathogen. In this study, we probed into the features of VNTRs in the  Y. pestis  genome and developed a simple hierarchical genotyping system based on optimized VNTR loci.    Methodology/Principal Findings  Capillary electrophoresis was used in this study for multi-locus VNTR analysis (MLVA) in 956  Y. pestis  strains. The general features and genetic diversities of 88 VNTR loci in  Y. pestis  were analyzed with BioNumerics, and a “14+12” loci-based hierarchical genotyping system, which is compatible with single nucleotide polymorphism-based phylogenic analysis, was established.    Conclusions/Significance  Appropriate selection of target loci reduces the impact of homoplasies caused by the rapid mutation rates of VNTR loci. The optimized “14+12” loci are highly discriminative in genotyping and source-tracing  Y. pestis  for molecular epidemiological or microbial forensic investigations with less time and lower cost. An MLVA genotyping datasets of representative strains will improve future research on the source-tracing and microevolution of  Y. pestis.

Features of Variable Number of Tandem Repeats in Yersinia pestis and the Development of a Hierarchical Genotyping Scheme