| Summary: | Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus <i>Bartonella</i> are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any <i>Bartonella</i> species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (<i>Sciurus vulgaris</i>), European hedgehog (<i>Erinaceus europaeus</i>) and Northern white-breasted hedgehog (<i>Erinaceus roumanicus</i>), were sampled and tested for <i>Bartonella</i> presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of <i>Bartonella</i> spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for <i>Bartonella</i> DNA detection. The wide spectrum of <i>Bartonella</i> spp. that were identified includes three species with previously validated zoonotic potential, <i>B. grahamii</i>, <i>B. melophagi</i> and <i>B. washoensis</i>, accompanied by ‘<i>Candidatus</i> B. rudakovii’ and two putative novel species, <i>Bartonella</i> sp. ERIN and <i>Bartonella</i> sp. SCIER.
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