Bovine anaplasmosis is a tick-borne disease in cattle which is mainly caused by Anaplasma marginale and Anaplasma centrale. It poses significant economic burdens and threat on livestock industries worldwide. This study aimed to identify Anaplasma spp. infecting the commercial Mafriwal cattle in Johor, Malaysia and investigate their phylogenetic relationship in the population. In this study, polymerase chain reaction (PCR) targeting the MSP4 gene for A. marginale and the 16s rRNA gene for A. centrale were performed. These assays were conducted on blood samples collected from 242 Mafriwal cattle. BLAST analysis and phylogenetic trees were constructed to analyze the genetic relationships between the Anaplasma spp. The results revealed 57.85% of the sampled population were infected with Anaplasma spp., 21.90% with A. marginale, 9.50% with A. centrale and 26.45% with both A. marginale and A. centrale. BLAST analysis showed 100% similarities between A. marginale sequences from this study and the sequence from a goat in Brazil. Similarly, A. centrale sequences were closely related to strains from tick vector, Rhipicephalus (Boophilus) microplus in Panama with 100% similarity. Phylogenetic analysis confirmed distinct clades for A. marginale and A. centrale, indicating genetic diversity and specific species differentiation. The findings highlight the endemicity of bovine anaplasmosis in Malaysian cattle populations and potential cross-border transmission routes. Moreover, this study provides the first report of A. centrale prevalence in Malaysia, emphasizing the importance of ongoing surveillance and management efforts. Understanding the genetic diversity and species differentiation of these pathogens is crucial for designing effective control strategies and vaccine development. In conclusion, this study enhances our understanding of the prevalence and genetic dynamics of bovine anaplasmosis among Mafriwal cattle in its largest population in Malaysia for better diagnosis and effective control measures.