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Tick-borne maladies of canines are increasing in sub-tropical and tropical areas around the globe. Theileriosis is an emerging issue in canines, especially dogs but the data regarding the disease prevalence in Asian countries is scarce. Therefore, the current study was designed to check the molecular prevalence of Theileria spp. in dogs of Lahore, Pakistan. A total of 102 dog blood samples were initially screened by microscopy and later on by PCR. The results revealed that 10.78% (11/102) and 6.86% (7/102) of pet dogs were found positive by PCR and microscopy, respectively. The phylogenetic analysis showed the sequences resemblance up to 98-99% with cytb gene fragments of Theileria annulata isolates from Iran, India, Turkey, Sudan, China, and Tunisia. Assumed risk factors revealed a significant (P<0.05) association of house hygiene, tick infestation, and tick prevention applications with the disease dynamics. The infected animals also showed a significant decrease (P<0.05) in values of red blood cells (RBCs), hemoglobin level, PCV, and platelet count. This is the first report regarding the molecular evidence of T. annulata infection in dogs of Pakistan. This study will help control the increasing tick-borne maladies of dog population in Pakistan.