Theileria parva is a haemoprotozoan parasite that causes fatal lymphoproliferative disease of cattle, East Coast fever (ECF), transmitted by Rhipicephalus appendiculatus tick. There are many stocks of T. parva differing in various features. Immunity elicited by immunization is stock specific and therefore, it is appropriate to characterize T. parva to find most relevant stocks for inclusion in the vaccine. This study determined molecular epidemiology of T. parva parasite in Eastern Zone of Tanzania by conventional polymerase chain reaction (PCR) and semi-nested PCR to amplify antigenic genes coding for sporozoite surface protein (p67) and piroplasm proteins (p104) respectively using DNA extracted from cattle blood samples naturally infected with T. parva. Restriction fragments length polymorphism (RFLP) profiles of p104 were generated by Arthrobacter luteus I (Alu I) restriction enzyme. Results demonstrated two types of p67 bands, 750 bp and 950 bp. Profiles for p104 PCR-RFLP revealed 5 types of stocks; the first and the second stocks produced PCR-RFLP profiles identical to T. parva Katete and T. parva Muguga. The p104 PCR-RFLP profiles of the rest three stocks were distinct novel variant stocks never reported in previous studies. The significance of the different p67 alleles and the novel variants stocks detected by p104 PCR-RFLP in the epidemiology of theileriosis in Eastern Zone of Tanzania and the key aspects for vaccine developement is discussed.