Background: Theileria annulata, a tick-borne apicomplexan parasite, causes tropical theileriosis in cattle, leading to significant economic losses in endemic regions. In our previous study, T. annulata was detected in various bovine blood samples via sequencing, despite negative results from conventional species-specific PCR assays, suggesting potential sequence variation in diagnostic target regions. Building upon this observation, the present study aimed to investigate the genetic diversity and population structure of T. annulata isolates from Türkiye by analyzing two molecular markers with differing levels of variability: the conserved 18 S rRNA gene and the polymorphic Tams1 gene. Results: A total of 36 bovine blood DNA samples previously confirmed as T. annulata-positive were analyzed using PCR and Sanger sequencing. All 36 sequences from the 18 S rRNA gene (717 bp) were identical, forming a single haplotype, and indicating strong conservation of this locus. In contrast, the Tams1 gene (584 bp) yielded 27 distinct haplotypes among 34 sequences, demonstrating high haplotype diversity (Hd = 0.988) and moderate nucleotide diversity (π = 0.0307). Neutrality tests revealed a significantly positive Tajima’s D (2.04089; P < 0.05) and a strongly negative Fu’s Fs (–5.685; P = 0.002) for the Tams1 region, indicating the presence of balancing selection and potential population expansion. Phylogenetic and haplotype network analyses further demonstrated significant genetic divergence within the Tams1 gene region, suggesting the presence of multiple divergent lineages and localized genetic structuring among isolates. Conclusion: This study represents one of the few sequence-based efforts to characterize the genetic structure of T. annulata in Türkiye using both conserved and polymorphic molecular markers. The findings reveal substantial genetic diversity within the Tams1 gene and underscore the limitations of relying solely on conserved targets for molecular diagnostics. These results suggest that regional variation in genetic diversity may influence diagnostic sensitivity, thereby supporting the potential value of incorporating polymorphic markers into surveillance strategies to improve epidemiological monitoring and control efforts.