A study was designed to improve understanding of the genetics of Theileria annulata populations in sympatric cattle and Asian buffalo (Bubalus bubalus). The study was undertaken in the Punjab province of Pakistan, where the prevalence of tropical theileriosis is high. Parasite materials were collected from infected animals in defined regions, where cattle and Asian buffalo are kept together. Six satellite DNA markers and a mitochondrial cytochrome b marker were used to explore the multiplicity of T. annulata infection and patterns of emergence and spread of different parasite genotypes. The results show differences in the numbers of unique satellite locus alleles, suggesting that T. annulata is genetically more diverse in cattle- than in buffalo-derived populations. Heterozygosity (H) indices based on satellite and cytochrome b loci data show high levels of genetic diversity among the cattle- and buffalo-derived T. annulata populations. When considered in the context of high parasite transmission rates and frequent animal movements between different regions, the predominance of multiple T. annulata genotypes and multiple introductions of infection may have practical implications for the spread of parasite genetic adaptations; such as those conferring vaccine cross-protection against different strains affecting cattle and Asian buffalo, or resistance to antiprotozoal drugs.Copyright © 2020 Elsevier GmbH. All rights reserved.