Hyalomma anatolicum (H. anatolicum) and Rhipicephalus microplus (R. microplus) are the major tick species affecting cattle populations around the world, especially in Pakistan. Climate change has led to an increase in their prevalence worldwide.  Field veterinarians reported a high prevalence of ticks during winter in district Chakwal of Pakistan. The present study was conducted in two different ecological zones to see the variation in tick infestation among different ecological zones. A total of 740 samples of ticks were collected from two selected districts. Tick identification was done under the stereomicroscope. Polymerase chain reaction (PCR) was performed using selected cytochrome C oxidase subunit 1 (COI) gene marker and phylogenetic analysis was also performed after sequencing. The overall prevalence of tick infestation in cattle was 69.46%. A significantly higher prevalence (P<0.05) in district Chakwal (80.54%) was observed than in district Faisalabad (58.38%). There was a significant difference in tick species between the two regions. In Faisalabad, H. anatolicum was the dominant species, with a prevalence of 72.69% whereas in Chakwal, R. microplus was more common, with a prevalence of 66.10%. Despite the colder winter temperatures in Chakwal, ticks were still found to have a prevalence of 19.38%, whereas in Faisalabad, their prevalence was nearly zero during winter. The association of different factors like gender, breed, and housing (walls, floor, grazing, and acaricide) was found to be statistically significant (P<0.05) for tick prevalence. In phylogenetic analysis, no variation in sub-species level was noted, ticks in both districts are 99.4% identical. This study will help in developing regional tick control programs and finding alternate tick control methods like immunological tick control. These results could be useful in the development of integrated tick and tick-born disease control strategies in Pakistan.

To Cite This Article: Ahmad M, Sindhu ZD, Zafar MA and Saqib M, 2025. Prevalence, Risk Factors, and Molecular Characterization of Hard Ticks in Two Diverse Agro-Ecological Zones of Punjab, Pakistan. Pak Vet J, 45(1): 336-343. http://dx.doi.org/10.29261/pakvetj/2025.128