Theileria are tick-transmitted, haemoprotozoan parasites that infect wild and domestic small ruminants in the tropical and subtropical regions of the world and cause an economically important disease (theileriosis) in domestic livestock. Theileria lestoquardi (T. hirci) and T. china 1 are considered highly pathogenic to small ruminants especially sheep. In contrast to the conventional methods, the use of molecular techniques allows specific and sensitive detection as well as differentiation of Theileria species. The main aim of this study is to use polymerase chain reaction (PCR)-based methods for detection and identification of Theileria species in sheep in two regions in Oman, Al Dakhliyah and Al-Batinah. The study examined polymorphism of 18S rRNA gene by using restriction fragment length polymorphism (RFLP) to differentiate between Theileria species. The sensitivity and specificity of this method in detection of Theileria parasites was assessed. In addition, the prevalence of different Theileria species was examined in two sites in Oman and the phylogenetic relationship of Theileria in Oman with those in other areas was determined. Using PCR-RFLP assay, 62% out of 231 examined sheep were found to be infected with Theileria species. The sensitivity of nested PCR for detection of Theileria species was 10-4% parasitemia, this is equivalent to approximately 1 parasite per 1 ul blood. Eighty eight (61%), 17 (12%) and 39 (27%) of the infections comprised of T. ovis, T. lestoquardi and mixed infection of T. ovis, T. lestoquardi and T. annulata, respectively. The prevalence of Theileria parasites in Al- Dakhliyah and Al-Batinah was 78 (54%) and 66 (46%) of 144 samples, respectively. T. ovis was the dominant parasite in both Al Dakhliyah (56%) and Al-Batinah (45%). Phylogenetic analysis showed that T. ovis in Oman is close to T. ovis in Spain, Sudan, Turkey, Croatia, Italy, Iran and France, however, T. lestoquardi is distanced from T. lestoquardi in Sudan and Iran. In conclusion, this is the first comprehensive molecular study in which different Theileria species have been identified and their distribution in different regions in Oman was asessed. The findings indicated that sheep in Oman are at a great risk of Theileria infection.