Yersinia pestis whole-genome DNA microarrays were developed to perform genomic comparison of a collection of live plague vaccines. By using the genomic DNA to probe the DNA microarrays, we detected dozens of deletions and amplifications of the genomic regions in the 19 vaccine strains analyzed. The revealed genomic differences within the vaccine strains of different origins provide us an unprecedented opportunity to understand the molecular background of the variability of the immunogenic and protective potency of plague live vaccine. The whole-genome DNA microarray also provides an ideal tool to perform the pre-evaluation of a vaccine strain for its high throughput to determine the genomic features essential or unallowable for the live vaccines.