In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.