The present study determined the prevalence, hematological findings and genetic diversity of Bartonella spp. in domestic cats from Valdivia, Southern Chile. A complete blood count and nuoG gene real-time quantitative PCR (qPCR) for Bartonella spp. were performed in 370 blood samples from cats in Valdivia, Southern Chile. nuoG qPCR-positive samples were submitted to conventional PCR for the gltA gene and sequencing for species differentiation and phylogenetic analysis. Alignment of gltA gene was used to calculate the nucleotide diversity, polymorphic level, number of variable sites and average number of nucleotide differences. Bartonella DNA prevalence in cats was 18·1% (67/370). Twenty-nine samples were sequenced with 62·0% (18/29) identified as Bartonella henselae, 34·4% (10/29) as Bartonella clarridgeiae, and 3·4% (1/29) as Bartonella koehlerae. Bartonella-positive cats had low DNA bacterial loads and their hematological parameters varied minimally. Each Bartonella species from Chile clustered together and with other Bartonella spp. described in cats worldwide. Bartonella henselae and B. clarridgeiae showed a low number of variable sites, haplotypes and nucleotide diversity. Bartonella clarridgeiae and B. koehlerae are reported for the first time in cats from Chile and South America, respectively.