The study was conducted to assess the prevalence of Theileria infection in tick vectors collected from healthy animals to record the natural infection level of theilerial parasite in the field condition. A total of 156 male and 110 semi-engorged female of Hyalomma anatolicum anatolicum were collected from cattle and buffaloes of two different agro climatic zones of Punjab, dissected and their salivary glands were stained with Methyl Green Pyronin (MGP) stain. The prevalence, intensity and abundance of Theileria annulata infection were higher in female ticks (15.45%, 5.08 and 32.88, respectively) than male ticks (8.97%, 1.69 and 18.86, respectively). The ticks collected from cattle had a higher prevalence, abundance and intensity (15.15%, 5.38 and 35.53, respectively) of T. annulata infection than the ticks collected from buffaloes (9.58%, 1.74 and 18.13, respectively). Hot and dry climate favored the development of T. annulata sporozoites in H. a. anatolicum ticks as the prevalence, abundance and intensity of T. annulata infection were higher in ticks collected from Western semi arid zone of Punjab as compared to those collected from the central plain zone with hot and humid climate.