Aims: The aim of this study was to isolate and identify potentially pathogenic strains of Yersinia enterocolitica in water samples collected from the upstream section of the Drwezca River in Poland. Methods and Results: Thirty-nine water samples were collected. Strains were isolated, identified with the use of the API (R) 20E test kit (Biomerieux, Marcy l'Etoile, France) at 37 degrees C, serotyped and subjected to a molecular analysis. Multiplex PCR was carried out to amplify three virulence genes: ail, ystA and ystB. Fragments of ail and ystA genes were not identified in the genetic material of the analysed strains. The ystB gene was identified in four strains. Yersinia enterocolitica strains of biotype 1A, which contain the ystB gene, may cause gastrointestinal problems. Conclusions: In our study, Y. enterocolitica strains of biotype 1A/ystB with serotypes 0 : 3, 0 : 5 and 0 : 8 were identified in samples collected from the Drwezca River which flows through the areas protected by Natura 2000, one of the largest networks of nature conservation areas in the European Union. The presence of Y. enterocolitica in the Drwezca River indicates that the analysed bacteria colonize natural water bodies. Significance and Impact of the Study: Most research focuses on food or sewage as a source of Y. enterocolitica infections. Little is known about the occurrence of this pathogen in natural waters. Our results show that natural waters are also a potential threat to human and animal health.