Background and Aim: Bartonella spp. are Gram-negative zoonotic bacteria that are transmitted to humans by several types of animal hosts, including rodents. Several studies have been conducted on the prevalence of Bartonella infections in rodents. However, the risk of rodent-associated Bartonella spp. infection in humans remains unclear. This study aimed to estimate the prevalence and genetic heterogeneity of Bartonella spp. in rodents and shrews from nine provinces of Thailand using culture and molecular techniques. Materials and Methods: A total of 860 blood samples from rodents and shrews across nine provinces of Thailand were collected from January 2013 to June 2016. Bartonella spp. were isolated from all samples using conventional culture techniques and polymerase chain reaction. Phylogenetic tree analysis was used to align the Bartonella sequences obtained from this study. Results: The prevalence of Bartonella spp. in rodents and shrews was 11.5% (99/860, 95% confidence interval: 9.3813.64%). The following nine species of Bartonella were detected: Bartonella tribocorum, Bartonella rattimassiliensis, Bartonella queenslandensis, Bartonella elizabethae, Bartonella chanthaburi spp. nov., Bartonellasatun spp. nov., Bartonella coopersplainsensis, Bartonella ranong spp. nov., and Bartonella henselae. The prevalence of Bartonella- positive animals differed significantly among provinces. Conclusion: To the best of our knowledge, the three novel Bartonella spp. isolated from rodents and shrews across Thailand were detected for the first time in this study. Further studies on the epidemiology of Bartonella infection in rodents and its interaction with human health should be conducted in accordance with the Thai government's "One Health" approach to humans, animals, and the environment. Keywords: Bartonella spp., phylogenetic analysis, polymerase chain reaction, rodents.