Canine monocytic ehrlichiosis is a highly prevalent disease in Brazil, where the genetic diversity of Ehrlichia canis remains undefined. In this study, we used the TRP36 gene to examine the genetic diversity of E. canis strains from naturally infected dogs residing in five distinct geographic regions in Brazil. E. canis DNA was detected in 82/126 (65%) dogs by dsb-specific PCR and E. canis was isolated in cell culture from 13 dogs. Sequences obtained from dsb genes amplified from the isolates were identical to the US E. canis strain. An extended molecular characterization based on the TRP36 gene identified two major genogroups based on differences among eight isolates. Isolates with tandem repeat amino acid sequence (TEDSVSAPA) identical to the previously reported TRP36 sequence were found in the midwest, northeast and southeast regions of Brazil, and classified into the US genogroup. A novel Brazilian genotype with a different tandem repeat sequence (ASVVPEAE) was also identified in midwest, northern and southern regions. Similarity in the N-terminal sequence of a US genogroup member with the Brazilian genogroup suggested that genomic recombination between the two genogroups may have occurred. Other subtypes within the Brazilian genogroup were also identified using C-terminal amino acid divergence. We identified two distinct major Brazilian genogroups and several subtypes based on analysis of TRP36, and such information will be useful for further genotyping and possible associations with disease severity, understanding of the genetic and antigenic variability of E. canis, and for developing strain-specific vaccines and diagnostic methods based on TRP36.