Objective To investigate clinicopathological, bacteriological and pathological aspects of an experimental infection with Yersinia pseudotuberculosis (Y. pseudotuberculosis) in hares to verify the efficacy of serology for the in vivo diagnosis. Moreover, the pathogenicity of two Y. pseudotuberculosis strains was investigated in order to detect potential differences. Methods Twelve European brown hares (Lepus europaeus, Pallas) were experimentally infected per os and via conjunctival mucosae with Y. pseudotuberculosis: six subjects were infected with a strain isolated from a naturally infected hare (YpH) and six subjects with a strain isolated from a naturally infected rabbit (YpR). Two hares were used as negative controls. All animals were subjected to clinical, bacteriological and serological examinations during 9 weeks following the infection and, at the end of the control period, subjects still alive were euthanized and submitted to a complete post mortem examination. Results All faecal samples collected during the control period were positive for bacteriological examinations and to a PCR for the inv gene of Y. pseudotuberculosis, while only one YpH-infected hare showed a positive haemocultures. From the 2nd to the 9th week post infection (pi), serological analysis revealed specific antibodies with titers ranging from 1:10 to 1:160 in all YpH-infected and two YpR-infected subjects. All the YpH-infected and two YpR-infected hares scored positive for Y. pseudotuberculosis by means of bacteriological investigations. Grossly, suppurative multifocal lesions were detected in liver, spleen, kidney and sub-mandibular lymph nodes in both YpH- and YpR-infected hares and confirmed with histopathology. Pulmonary lesions were observed only in YpH-infected subjects. Immunohistochemistry confirmed the presence of bacterial antigen in all infected animals. Conclusion Results of this study revealed that YpH strain is more pathogenic for hares than the YpR strain; moreover the serological test performed in this study could be used for the diagnosis of pseudotuberculosis in hares, whereas post mortem diagnosis should be confirmed by means of bacteriological examination, PCR, histopathology and immunohistochemistry.