Abstract Objective—To determine whether Haemobartonella canis and Mycoplasma haemofelis (formerly known as H felis [large form]) can be differentiated by use of comparative analysis of gene sequences. Sample Population—Blood samples obtained from 3 dogs infected with H canis and 2 cats infected with M haemofelis. Procedure—The partial 16S rDNA and ribonuclease P RNA (RNase P) genes were amplified, cloned, and sequenced in blood samples obtained from H canis-infected dogs and M haemofelis-infected cats. The DNA sequences were subjected to comparative analysis. Results—The 16S rDNA sequences of H canis and M haemofelis were nearly identical (homology of 99.3 to 99.7%). In contrast, RNase P gene sequences had a lower degree of sequence homology between the 2 organisms (94.3 to 95.5%). Conclusions and Clinical Relevance—Haemobartonella canis and M haemofelis are not identical organisms. Molecular differentiation of H canis and M haemofelis is more clearly evident by use of comparative analysis of RNase P gene sequences than by comparative analysis of 16S rDNA gene sequences. (Am J Vet Res 2002;63:1385–1388)