Objective To explore the prevalence of Bartonella in small mammals in some regions of Inner Mongolia, and provide scientific basis for the control and prevention of Bartonella infection in population of small mammals in Inner Mongolia. Methods Night trapping methods were applied to capture small mammals in different regions of Inner Mongolia in 2012 and 2013. Livers and spleens were collected by aseptic technique, and then the tissues were subjected to PCR to test the infection of Bartonella. Afterward, the positive PCR products were sequenced, and the nucleotide sequences were submitted to GenBank accompanying further comparison and analysis. To identify the species of Bartonella, 30 livers and 30 spleens samples were cultivated for Bartonella bacteria, and then extract DNA of suspected strains. Subsequently, sequence the 379 bp fragment of gltA gene and make a phylogenetic analysis. In addition, the prevalence of Bartonella was analyzed based on different species and viscera. Results The small mammals were captured in Erenhot, Xilin Gol, Inner Mongolia in 2012, and positive Bartonella was cultured from 8 species of small mammals corresponding positive rate 56.41%(66/117). The positive rate of liver DNA amplification performed by direct PCR was 57.26% (67/117). There was no marked significance between them (P=0.945). 21 of the 30 liver samples were positive for Bartonella while 13 of the 30 spleens samples were positive. There was no statistic significance between them (P=0.331). 86 small mammals were captured in 2013, including 13 species of rodents, and 33 stains of Bartonella were isolated from these rodents with infection rate 38.37%(33/86). As to the positive infection rate of Bartonella, the highest infection rate was Spermophilus dauricus (75.00%), followed by Allactaga sibirica (71.43%) and Lasiopodomys brandti (64.29%). There were no significant differences in Bartonella prevalence among three species of small mammals (P=0.883). Total of 4 strains of Bartonella were confirmed from small mammals in some regions of Inner Mongolia via DNA sequencing in the rodents: B. jaculi, B. grahamii, B. washoensis, B. vinsonii. Conclusion Bartonella is widespread in small mammals in Inner Mongolia, which increase the risk to human population. Sequencing analysis shows the diversity of genotype of Bartonella. Our study provides a solid foundation for the further research on Bartonella in Inner Mongolia and other regions in northern China.