We developed and optimized a fluorogenic 5′ nuclease polymerase chain reaction assay, which is specific for the 16S rRNA present in the majority of Yersinia species. The optimized assay uses the reporter dye VIC and the quencher dye TAMRA. The assay amplified all species of Yersinia, except Y. ruckeri, which is fish pathogen. The assay detects five picograms of Yersinia DNA. The assay was used to screen tonsil swab (n = 533) collected during the National Animal Health Monitoring System (NAHMS) Swine 2000 study. Tonsil swabs were received from 101 farms from 17 states and shipped to National Animal Disease Center for processing. Of the 553 tonsil samples screened, 61.8% (342) were positive for the 16S rRNA gene of Yersinia.