The present study was carried out to determine the prevalence of Theileria lestoquardi from two districts of Khyber Pukhtoon Khwa (Kohat and Peshawar) in Pakistan and also to report the risk factors associated with the spread of ovine theileriosis. A total of 165 blood samples were collected from sheep (N = 44) and goats (N = 121) from randomly selected herds. Data on the characteristics of animals and the herds were collected through questionnaires. Five (3%) out of total 165 samples produced 730 base pairs DNA fragment, through PCR amplification of 18S SSU rRNA gene, specific for T. lestoquardi. All positive samples were from district Kohat while samples from Peshawar were found negative for this parasite. Statistical analysis indicated a significant association (P = 0.005) between sampling site and prevalence of T. lestoquardi. It was observed that presence of tick on the ruminant (P = 0.0007) and the dogs associated with the herd (P = 0.001) were highly significant risk factor for the spread of ovine theileriosis. It was also observed that mixed herds (containing both sheep and goats) were more prone to the parasite. We have concluded that PCR is a sensitive and reliable diagnostic tool for detection of T. lestoquardi in blood samples of small ruminants and can be used for the prophylactic screening and treatment of this blood parasite in order to increase the live stock production in Pakistan.