Blood-borne parasites, including Babesia spp., Theileria spp., and Anaplasma spp., pose significant threats to buffalo health and productivity, highlighting the need for continuous epidemiological surveillance in endemic regions such as Thailand. This study aimed to investigate the molecular prevalence of hemoparasites and characterize the genetic diversity of Theileria orientalis in swamp buffaloes from Northeast Thailand. A total of 176 blood samples were analyzed using genus- and species-specific PCR assays. Samples positive for T. orientalis were subjected to DNA sequencing of the MPSP gene, followed by haplotype and phylogenetic analyses. Overall, 19.3% (95% CI: 13.8–25.9) of samples were positive for at least one hemoparasite. The prevalence of T. orientalis, Babesia bigemina, and B. bovis was 15.9%, 3.4%, and 2.3%, respectively. Notably, all B. bovis infections occurred as co-infections with T. orientalis. Anaplasma spp. showed the lowest prevalence (1.7%), comprising A. marginale 0.6% and A. platys 1.1%. Sequence analysis of T. orientalis revealed significant genetic diversity within the population. Subsequent phylogenetic and pairwise distance analyses identified four distinct genotypes including Type 7, which was found to be predominant, followed by Type 5, Type N2 and Type 4. This study provides molecular evidence of hemoparasite infections and highlights the complex genetic diversity of T. orientalis in Thai water buffaloes. The co-circulation of multiple genotypes and co-infections underscore the need for improved epidemiological monitoring and targeted control strategies in the region.