Background: Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when common ecological niche is shared in the presence of a competent vector. Method: To investigate piroplasm diversity in wildlife and cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using illumina MiSeq sequencing platform and amplicon sequence variant (ASV) based bioinformatics pipeline to generate high resolution data which discriminate sequences down to a single nucleotide difference. Results: A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to cattle population in the wildlife-livestock interface.Conclusion: We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.