Background Bartonella spp. are vector-borne pathogens transmitted to humans via blood-sucking arthropods. Rodents such as the black rat (Rattus rattus) and Norway rat (R. norvegicus) are thought to be the main reservoirs. An infection with rodent-associated Bartonella spp. may cause severe symptoms in humans such as endocarditis and neuroretinitis. The current knowledge of Bartonella prevalence in rats from western Europe is scarce. Methods Rats and a few other rodent by-catches were trapped in the context of a rodenticide resistance study at different sites in Flanders, Belgium. During dissection, biometric data were collected, and spleen tissues were taken. DNA was extracted from spleen samples and tested for Bartonella spp. by conventional generic polymerase chain reaction (PCR). To determine the Bartonella species, a selected number of amplicons were sequenced and compared with GenBank entries. Results In total, 1123 rodents were trapped. The predominate species was R. norvegicus (99.64%). Other rodents trapped included: two water voles (Arvicola amphibius, 0.18%); one colour rat (R. norvegicus forma domestica, 0.09%); and one muskrat (Ondatra zibethicus, 0.09%). PCR analysis of 1097 rodents resulted in 410 (37.37%, 95% CI: 34.50–40.31%) Bartonella spp. DNA-positive samples. Bartonella tribocorum (94.68%, 95% CI: 88.02–98.25%) was the most frequently detected Bartonella species, followed by B. grahamii (3.19%, 95% CI: 0.66–9.04%) and B. doshiae (1.06%, 95% CI: 0.03–5.79%). An uncultured Bartonella species occurred in one water vole (1.06%, 95% CI: 0.03–5.79%). There was a significantly higher Bartonella prevalence in older rats compared to juveniles and a significant difference in Bartonella prevalence concerning the localisation of trapping sites. In contrast, there was no statistically significant difference in Bartonella prevalence regarding sex, degree of urbanisation and season. Conclusions Based on the high prevalence found, we conclude that the Norway rat seems to be a key reservoir host for zoonotic ...