Thirty bartonella strains were isolated from the blood of black-tailed prairie dogs (Cynomys ludovicianus) from Boulder County, Colorado, USA. The bacteria appeared as small, fastidious, aerobic, Gram-negative rods. The partial sequences of the citrate synthase gene (gltA) demonstrated five unique genetic variants. Phylogenetic analysis based on sequences of gltA, 16S rRNA, rpoB, ftsZ, and ribC showed that the black-tailed prairie dog-related Bartonella variants comprise a distinct monophyletic clade that is closely related to Bartonella washoensis, a species isolated from a human patient and subsequently from ground squirrels. These variants, however, are grouped together in 100% of the bootstrapped trees. These variants were not found in other small mammals trapped during the same study, showing some evidence of host specificity. We believe that the group being described here is typical of the black-tailed prairie dog. We propose to name the bacteria Candidatus Bartonella washoensis subsp. cynomysii. The type strain is CL8606co(T)(=ATCC BAA-1342(T) = CCUG 53213(T)), which is the representative isolate of the dominant variant of the characterized group.