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Abstract Background Tick borne diseases impinge cattle worldwide causing mortality and resulting in huge economic losses. Theileriosis is one of the important tick borne diseases mainly caused by Theileria annulata and one of the commonly occurring infections among the livestock. T. annulata causes immense loss to the livestock industry and therefore, efficacious eradication and control strategies are needed for the control of the disease. Genetic diversity among T. annulata parasites is another important aspect which is overlooked in India. Thus, the present study aims to evaluate the prevalence along with genetic diversity and phylogeny of the prevailing T. annulata population of India. Methods Genomic DNA was extracted from cattle blood samples (n = 862) from different regions of Andhra Pradesh. Molecular diagnosis using T. annulata 18S rRNA based PCR was performed to detect parasites in cattle. Further, 18S rRNA gene was cloned and sequenced to determine similarity and diversity from the known T. annulata sequences. Results We observed an overall prevalence rate of 32.40 % T. annulata infection in Andhra Pradesh based on PCR assay. The sequence analysis revealed novel genotypes among the T. annulata strains from India. Thirteen strains showed closed proximity with a strain from China whereas one Indian strain showed similarity with a South African strain [Theileria sp (buffalo)] based on phylogenetic analysis. Nucleotide heterogeneity of the 18S rRNA sequence among the strains examined varied from 0.1 to 8.6 % when compared with the published strains. Conclusion The present study provides us with the molecular prevalence of theileriosis, and will support the accomplishment of actions or in design of strategy to control theileriosis transmission to cattle. Additionally, it highlights the emergence of strains with novel genotypes from India.