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Bovine anaplasmosis is a serious tick-borne disease that is responsible for economic loss worldwide. The major surface proteins (MSPs), encoded by msp1to msp5genes of Anaplasma marginale, play an important role in host-pathogen and tick-pathogen interactions. These markers have been used for genetic characterization and phylogenetic studies. Despite domestic reports concerning suspected outbreaks of anaplasmosis in Thailand, genetic analysis of A. marginalein the country remains largely limited. Therefore, we aim to investigate the infection rate of the rickettsia organism in the Anaplasmataceae family throughout five regions of Thailand and to further characterize the key genetic markers: msp1a, msp2, and msp5of A. marginale. From 2016 to 2021, we collected a total of 384 cattle blood samples across 18 provinces. Overall, the infection rate of the rickettsia organism in the Anaplasmataceae family was 46.1%. Over 65% of the positive samples were confirmed as A. marginale. We successfully obtained a total of 138 A. marginale msp1a(38), msp2(79), and msp5(21) sequences from all regions of the country. The msp1aand msp2genes exhibit a high degree of genetic diversity, while the msp5gene is highly conserved among the Thai isolates. Our findings regarding msp1acorroborated the genetic heterogeneity of A. marginalestrains in endemic regions worldwide. Additionally, we found multiple novel variants for the first time in the current nationwide survey. We found 45 tandem repeat characters of the msp1asequence. Among them, 24 characters were not shared with other countries. Collectively, we expanded the extent of genetic diversity in key markers; msp1aand msp2genes, and further confirmed the previous finding that msp5was highly conserved. The msp1aand msp2genes could be useful for the surveillance of newly introduced strains. The current data may also be useful in designing a vaccine containing potential epitopes of different antigens in the future.