Theileriosis is a tick-borne disease caused by the protozoan parasite of the genus Theileria, and is transmitted by ticks of several different genera. The pathogenic parasite species cause severe morbidity and high rates of mortality resulting in considerable economic losses. In Oman both bovine and ovine theileriosis are widespread, Limited studies have highlighted the importance of ovine theileriosis, and reported high prevalence of Theileria species in two governates, with a large number of animals carrying more than one species. In addition, preliminary phylogenetic analysis suggested that Theileria lestoguardi in Oinan is relatively distinct from other species in the region. Thus, the present study was conducted to a) determine the prevalence and distribution of Theileria species in different geographic regions across the country, b) assess risk factors for Theileria spp infection and c) elucidate evolutionary relationship of the most pathogenic ovine species (T. Lestoguardi) in Oman and countries in the region. A total of 2020 samples representing 10% of the main livestock population (cattle (n=447], sheep (n=592] and goats (n=981]) were collected from four governates, Ash Sharqiyah, Ad-Dhahira, Al-Batinah and Dhofar. Theileria species were identified using Polymerase Chain Reaction-Restriction fragment length polymorphism (PCR-RFLP), reverse line blot (RLB) and species-specific PCR. For phylogenetic analysis, 22 T. lestoquardi isolates from sheep were cloned, sequenced and analyzed together with other sequences, of T. lestoquardi from other countries available in GeneBank using BioEdit, MEGA, FaBox and Network programs. Detection and Theileria species identification of RLB and PCR-RFLP were found to be comparable. However, RLB detected more mixed species infection, than PCR-RFLP. Four Theileria different species were identified: T. annulata, T. lestoquardi, T. ovis and T. sp. OTI. The prevalence of Theileria spp differed significantly between different governates; Dhofar (36.1%) has the highest prevalence followed by Ad Dhahira (30.6%), Ash-Sharqiyah (28.6%) and Al-Batinah (19.9%). The prevalence of Theileria spp was considerably high in bovine (72.3%) and ovine (36.6%), compared to caprine (2.7%). Theileria annulata was the only species seen in bovine samples (72.3%), while all the four species were detected in small ruminants. In ovine 14.5% of the samples carried single infection of T. ovis, compared to 3% with T. lestoquardi infection and 0.2% positive with T. annulata. However, a large proportion of infected ovine (19%) carried mixed species infection of T. ovis and T. lestoguardi. Host, breed and gender were the main risk factors associated with Theileria spp. infection in Oman. Two hosts were at high risk of Theileria (avine and bovine), while caprine was not. Exotic breeds showed high susceptibility to infection compared to indigenous and cross breeds. Male ovine hosts showed significantly higher prevalence of Theileria parasite compared to females (p= 0.004). However, no significant differences in the prevalence of Theileria spp in different age groups was seen, although in bovine there was a trend of increased rates of infection as age increased. Sequence of analysis revealed higher diversity of Msl (23 nucleotides variation sites) compared to 18S rRNA gene (5 nucleotide variation sites). Nine distinct Mşl haplotypes were seen compared to 5 for 18S rRNA, Phylogenetic analysis of T. lestoquardi 18s rRNA genes revealed that the Oman's parasites were very similar to each other, and were closely related to parasites in Iran. Msl gene analysis support the finding from 18s rRNA, however, one Oman's lineage was found to be distanced from the others. Theileria lestoquardi was phylogenetically close to T. annulata and diverged from T. ovis based on 18S rRNA gene. In conclusion, the present study demonstrated that Oman is highly endemic for Theileria parasite and occurrence of the disease was high in different regions in the country. Two economically important hosts (cattle and sheep) are at high risk of Theileria infection, while goats, are proven to be resistant. Exotic-breed of cattle and sheep are highly susceptible to the infection than indigenous and cross animals. There is a trend of high susceptibility of cattle aged more than 3 years. The high prevalence of T. lestoquardi (the cause of ovine theileriosis) as mixed rather than single infection requires further investigation. Analysis of within host dynamics of mixed species infection may reveal factors that promote persistence and transmission of this parasite. Further sequence and phylogenetic analysis of T. ovis and T. annulata lineages in Oman would allow better understanding the evolution of the three parasites, in turn, this would lead to development of better control strategies based on vaccine or therapy.