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Three worldwide historical plague pandemics resulted in millions of deaths. <i>Yersinia pestis</i>, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of <i>Y. pestis</i> is important to prevent and control plague. However, the high similarity between <i>Y. pestis</i> and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the <i>Y. pestis</i> CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 <i>Y. pestis</i> strains and 572 strains of 20 species within the <i>Yersinia</i> genus. Altogether, 97 <i>Y. pestis</i>-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished <i>Y. pestis</i> from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR–fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by <i>Y. pestis</i> was significantly higher than that of non-<i>Y. pestis</i> (<i>p</i> < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.