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Background: Landscape anthropization and interaction between domestic and wild animals are the major contributing factors involved in the emergence of new pathogens in wild animals. Theileriosis is an emerging issue of wild ungulates, especially in the tropical and subtropical areas of the globe. Aims: The current study investigated the mouflon sheep for Theileria infection using molecular methods and hematological analysis. Methods: This study was conducted on a total of 103 captive wild mouflon sheep present in eight different recreational zoos, and wildlife parks in Punjab, Pakistan to investigate the genotypic prevalence of Theileria spp. by targeting 18S rRNA and molecular evidence for Theileria spillover between domestic and wild mouflon sheep by phylogenetic analysis. The association of assumed risk factors and the effect of Theileria spp. on various hematological parameters were also assessed. Results: The results depicted that Theileria spp. was prevalent in 8 (7.77%, CI 95%: 3.99-14.59%), and 11 (10.68%, CI 95%: 06.07-18.12%) animals based on microscopy, and PCR, respectively. The phylogenetic analysis of the 18S rRNA gene of Theileria spp. from mouflon revealed a close resemblance with T. annulata from domestic animals. The risk factor analysis revealed that tick infestation, enclosure hygiene, previous tick infestation history, and the presence of wooden logs in the enclosure were significantly (P<0.05) associated with the occurrence of Theileria spp. infection in the captive mouflon sheep of Pakistan. Furthermore, a significant reduction in blood parameters like PCV, RBCs count, Hb, and platelets was observed in Theileria-positive animals. Conclusion: This study is the first evidence at the molecular level to characterize the spillover of Theileria spp. between the captive wild mouflon sheep and domestic animals of Pakistan, and it will be useful in developing control strategies for emerging theileriosis in captive wild animals. [ABSTRACT FROM AUTHOR]