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The prevalence of Theileria spp. infecting sheep and goats were investigated in two cities and surroundings in northwest Saudi Arabia. Blood samples from 403 sheep and goats from Madina ( n = 201) and Tabuk ( n = 202) cities (177 from sheep and 226 from goats) were investigated. Blood samples were examined microscopically for the presence of intraerythrocytic bodies suggestive of Theileria as well as they were investigated using molecular techniques. DNA was extracted from blood and ticks and subjected to polymerase chain reaction amplification using specific primers. The primers used amplified a fragment of the 18S rRNA region (450 bp) targeting the hypervariable region IV. A total of 63 ticks belonging to five species were collected from sheep and goats for determination of their involvement of lifecycle of Theileria . Ticks were identified morphologically and confirmed molecularly utilizing cytochrome oxidase C subunit 1 gene (COXI) amplification. The results indicated that: microscopic examination revealed 24 (6%) of the samples investigated showed intraerythrocytic bodies suggestive of Theileria . Positive samples were only obtained from sheep whereas goats yielded negative results. A total of 33 (18.6%) sheep samples were positive for Theileria infection using polymerase chain reaction targeting the partial 18S rDNA and DNA sequencing. Theileria infection was more prevalent in animals that were less than 2 years of age compared with older animals. There was no difference in the prevalence of the infection between male and female sheep in both cities. All positive sheep were detected during the summer and none of the samples collected during the winter were positive. Phylogenetic analysis revealed that the sequences obtained from Theileria species reported in the present study grouped with sequences from Theileria ovis from different countries. Ticks were identified as Hyalomma dromedarii , Hyalomma, marginatum , Hyalomma impeltatum and Hyalomma anatolicum anatolicum . T. ovis DNA was detected from Hyalomma dromedarii and Hyalomma impeltatum suggesting that they are potential vectors of this piroplasm in sheep from Madina and Tabuk cities. This report is considered the first report of T. ovis infecting sheep from Madina and Tabuk, furthermore, it is the first report determining the vectors responsible for transmission of the infection in sheep in northwest Saudi Arabia. The data generated from this study will undoubtedly pave the way for the detection and control of ovine and caprine theileriosis in Madina and Tabuk regions., Competing Interests: The authors declare that they have no competing interests., (© 2024 Almahallawi et al.)