Bartonella is a widely distributed Gram-negative bacterium that includes species that are capable of causing illness in humans. Rodents represent one of the main reservoirs of zoonotic pathogens, and monitoring their populations can provide valuable insights into human health. We conducted a surveillance study of rodents from two north-western states of Mexico (Baja California and Chihuahua) to investigate the prevalence and genetic diversity of Bartonella by polymerase chain reaction (PCR) amplification and sequencing of the citrate synthase (gltA) gene. A total of 586 rodents belonging to 28 species were captured, and 408 were tested for Bartonella spp. The overall Bartonella spp. prevalence was 39.71%. The prevalence found in Chihuahua was higher (42.80%) than in Baja California (32.52%), and rodents such as Neotoma albigula, Neotoma mexicana, Peromyscus boylii, and Chaetodipus baileyi had the highest prevalence. The gltA sequences revealed seven genetic variants, some of which were obtained from Peromyscus and Dipodomys rodents and were associated with Bartonella species of human health concern, such as B. grahamii and B. vinsonii subsp. arupensis. In addition, a sequence obtained from a Peromyscus maniculatus was clustered with Candidatus Bartonella rudakovii, a previously unreported association. This study provides valuable data and new insight into the Bartonella-hosts interactions in rodent species in north-western Mexico.