East Coast Fever (ECF) is one of the most economically important tick-borne diseases of cattle in Eastern, Central, and Southern Africa, caused by the intracellular protozoan parasite Theileria parva (T. parva). This study investigated the prevalence and genetic diversity of T. parva populations in the eastern Democratic Republic of Congo (DRC) to inform immunization strategies against ECF. By employing PCR and DNA sequencing techniques, in conjunction with surveys assessing Knowledge and Practices, as well as conducting an immunization and field challenge trial, the findings suggest the existence of Rhipicephalus appendiculatus ticks in the provinces of South-Kivu, North Kivu, and Ituri. This underscores a possible threat of disease transmission in these areas. Molecular analyses uncovered diverse T. parva populations with varying antigenic profiles, challenging the assumption of uniformity despite Muguga cocktail-like appearances. Moreover, phylogenetic analyses suggested limited similarity between T. parva populations and the Muguga cocktail vaccine. Microsatellite analysis and field challenge trials supported the notion of multiple populations, highlighting the current vaccine\'s limitations against field strains. This study has the potential to significantly contribute to understanding T. parva dynamics in the region, emphasizing the complexities of vaccine strain selection and stressing the importance of continuous monitoring and adaptive control strategies in the face of evolving parasite populations.