The present study aimed to investigate the prevalence of cattle ticks and molecular identification of R. microplus in different ecological regions of North-Western Pakistan. A total of 1091 ticks were collected from 430 cattle followed by morphological identification of different species and confirmation of R. microplus using polymerase chain reaction. Overall, 335 (77.9%) cattle were infested with ticks. Grazing system, lack of acaricide application, and age (with 86.0%, 55.8%, and 85.7% prevalence rates, respectively) were significantly (P ≤ 0.05) associated with higher tick infestation. Microscopic examination detected three different species of ticks including R. microplus, Hyalomma anatolicum, and Hyalomma marginatum at prevalence rates of 47.8%, 41.8% and 10.4%, respectively. Rhipicephalus microplus identification was also confirmed by sequencing the second internal transcribed spacer (ITS2) and cytochrome oxidase I (COI) gene fragments. Phylogenetic analysis of ITS2 revealed a single clade of R. microplus and based on the COI nucleotide sequencing, R. microplus in the present study was assigned to clade C. This study showed a higher prevalence of cattle ticks in North-Western Pakistan and confirmed R. microplus as a major cattle tick species. These findings may be helpful in devising strategies for integrated control of ticks and tick-borne diseases in Northwestern Pakistan.