Abstract: Background and Objective: Knowledge of local isolates and strains is a prerequisite for development of both effective mass vaccination strategy and a suitable molecular marker based diagnostic tool. The pathogenesis of bovine tropical theileriosis (BTT) caused by Theileria annulata in susceptible ruminants is known to vary depending upon the isolate and strain involved. The present study was designed to characterize T. annulata isolates from Northern India using PCR-RFLP technique. Materials and Methods: In the present study, HSP 70 gene was amplified from 28 naturally infected calves found in BTT endemic semi arid zone of Northern India. The amplified product was then digested with two restrictions enzymes viz., Taq I and Alu I to find out the pattern of restriction digests so as to have an idea of various strains of T. annulata involved. Results: With Taq I enzyme, two restriction digestion products were noticed with molecular weights of 100 and 175 bp, respectively. So far as Alu I enzyme is concerned, three restriction digestion products with molecular weights of 60, 90 and 125 bp, respectively, were noticed. Conclusion: Monomorphic pattern of restriction digest was noticed in all 28 studied T. annulata isolates, for both the enzymes, suggesting of presence of single strain of parasite involved in the studied area.