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Abstract Background Yersinia enterocolitica is widespread within the humans, pigs and wild boars. The low isolation rate of Y. enterocolitica from food or environmental and clinical samples may be caused by limited sensitivity of culture methods. The main goal of present study was identification of presumptive Y. enterocolitica isolates using MALDI TOF MS. The identification of isolates may be difficult due to variability of bacterial strains in terms of biochemical characteristics. This work emphasizes the necessity of use of multiple methods for zoonotic Y. enterocolitica identification. Results Identification of Y. enterocolitica isolates was based on MALDI TOF MS, and verified by VITEK® 2 Compact and PCR. There were no discrepancies in identification of all human’ and pig’ isolates using MALDI TOF MS and VITEK® 2 Compact. However three isolates from wild boars were not decisively confirmed as Y. enterocolitica. MALDI TOF MS has identified the wild boar’ isolates designated as 3dz, 4dz, 8dz as Y. enterocolitica with a high score of matching with the reference spectra of MALDI Biotyper. In turn, VITEK® 2 Compact identified 3dz and 8dz as Y. kristensenii, and isolate 4dz as Y. enterocolitica. The PCR for Y. enterocolitica 16S rDNA for these three isolates was negative, but the 16S rDNA sequence analysis identified these isolates as Y. kristensenii (3dz, 4dz) and Y. pekkanenii (8dz). The wild boar’ isolates 3dz, 4dz and 8dz could not be classified using biotyping. The main bioserotype present within pigs and human faeces was 4/O:3. It has been shown that Y. enterocolitica 1B/O:8 can be isolated from human faeces using ITC/CIN culturing. Conclusion The results of our study indicate wild boars as a reservoir of new and atypical strains of Yersinia, for which protein and biochemical profiles are not included in the MALDI Biotyper or VITEK® 2 Compact databases. Pigs in the south-west Poland are the reservoir for pathogenic Y. enterocolitica strains. Four biochemical features included in VITEK® 2 Compact known to be common with Wauters scheme were shown to produce incompatible results, thus VITEK® 2 Compact cannot be applied in biotyping of Y. enterocolitica.