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<i>Bartonella</i> is an intracellular parasitic zoonotic pathogen that can infect animals and cause a variety of human diseases. This study investigates <i>Bartonella</i> prevalence in small mammals in Yunnan Province, China, focusing on tissue tropism. A total of 333 small mammals were sampled from thirteen species, three orders, four families, and four genera in Heqing and Gongshan Counties. Conventional PCR and real-time quantitative PCR (qPCR) were utilized for detection and quantification, followed by bioinformatic analysis of obtained DNA sequences. Results show a 31.5% detection rate, varying across species. Notably, <i>Apodemus chevrieri, Eothenomys eleusis, Niviventer fulvescens, Rattus tanezumi, Episoriculus leucops, Anourosorex squamipes</i>, and <i>Ochotona Thibetana</i> exhibited infection rates of 44.4%, 27.7%, 100.0%, 6.3%, 60.0%, 23.5%, and 22.2%, respectively. Genetic analysis identified thirty, ten, and five strains based on <i>ssrA</i>, <i>rpoB</i>, and <i>gltA</i> genes, with nucleotide identities ranging from 92.1% to 100.0%. <i>Bartonella</i> strains were assigned to <i>B. grahamii</i>, <i>B. rochalimae</i>, <i>B. sendai</i>, <i>B. koshimizu</i>, <i>B. phoceensis</i>, <i>B. taylorii</i>, and a new species identified in <i>Episoriculus leucops</i> (GS136). Analysis of the different tissues naturally infected by <i>Bartonella</i> species revealed varied copy numbers across different tissues, with the highest load in spleen tissue. These findings underscore <i>Bartonella</i>’s diverse species and host range in Yunnan Province, highlighting the presence of extensive tissue tropism in <i>Bartonella</i> species naturally infecting small mammalian tissues.