Ehrlichia canis is an obligate intracellular bacterium and the etiologic agent of canine monocytic ehrlichiosis, an infectious disease that affects dogs and is often associated with thrombocytopenia. This study aimed to analyze the genetic variability of E. canis in thrombocytopenic dogs from two mesoregions in the state of Rio de Janeiro, Brazil. We analyzed 404 blood samples from dogs with thrombocytopenia using the dsb, p28, and trp36 genes as molecular targets. The positivity rate was 21.04% (85/404) for the dsb and p28 genes, while the trp36 gene had a detection rate of 34.1% (29/85) in the positive samples. To analyze genetic diversity, 23 samples positive for the p28 gene and 29 samples based on trp36 results were sequenced. Phylogenetic analysis revealed high genetic diversity among the circulating E. canis strains in the mesoregions studied, with the identification of distinct genogroups, including the American and Brazilian genogroups. Analysis of the p28 gene revealed 17 distinct haplotypes (Hd = 0.9872) with region-specific clustering. The haplotype network mirrored the trp36-defined genogroups, suggesting genetic structuring associated with the American and Brazilian variants. It is concluded that there is high genetic diversity in the trp36 and p28 genes of E. canis strains circulating in dogs in the Metropolitan and south Fluminense mesoregions of Rio de Janeiro state, highlighting the importance of molecular surveillance to better understand the epidemiology of canine ehrlichiosis and the strains involved in the infection