Ectoparasites (insect and arachnids) are a worldwide problem, which are responsible for up to 80% losses to the livestock economy. Among these, the Ixodids (hard ticks) are the most important and also transmit diseases like babesiosis, theileriosis and anaplasmosis. The present study was aimed to identify prevalence and distribution of the hard tick species using morphological and molecular keys in large ruminants of Pothohar region of Pakistan. Morphological investigations of ticks from 967 cattle and 665 buffaloes revealed the genera (3) and species (13) of various Ixodid ticks mainly Rhipicephalus (R), Hyalomma (Hy), and Dermacentor (D) comprised of R. microplus, R. annulatus, R. australis, R. eversti, R. turanicus, R. decloratus, Hy. dromedarii, Hy. scupens, Hy. rufipes, Hy. truncatum, Hy. excavatum, Hy. detritum and D. atrosignatus. Tick invasion was statistically higher in cattle (53.48%) than buffaloes (40.70%). Among these 10 species identifications were confirmed through morphological identification, however, molecular characterization of tick analysis (using partial ITS2 gene) confirmed prevalence of R. microplus, Hy. detritum and D. atrosignatus. This study provides the basic genetic identification tool for investigating the tick species and their phylogenetic relationship in tick species commonly found in humans and animals. The precise identification unquestionably can play a dynamic role in employment of appropriate control measurements, as different genera/species cause different diseases in livestock. Different tick species identified on the basis of morphological features and molecular characterization, will add value to the existing knowledge in identification of ticks in the Pakistan, as well as helpful in various disease diagnosis spread due to these hard tick species.