<p>Hard ticks are important vectors of medical and veterinary pathogens. Accurate species identification is crucial for understanding the dynamics of tick-borne diseases, yet the presence of cryptic species, damaged specimens, or immature stages often limits morphological methods. This study investigated the genetic diversity and phylogenetic relationships of ticks in northwestern Iran. In total 283 ticks collected from domestic livestock across ten towns in Ardabil Province. Morphological identification was complemented by molecular analyses using mitochondrial COI and nuclear ITS2 markers. Phylogenetic trees were constructed using Maximum Likelihood. Genetic distances were estimated with the Kimura two-parameter (K2P) model. Morphological and molecular analyses identified three species: <i>R. sanguineus</i> (<i>n</i> = 132), <i>H. marginatum</i> (<i>n</i> = 80), and <i>D. marginatus</i> (<i>n</i> = 71). COI and ITS2 markers reliably discriminated species and revealed frequent misidentification between <i>R. sanguineus</i> and <i>R. turanicus</i>. ITS2 haplotypes of <i>R. sanguineus</i> matched populations from Italy and Israel, whereas COI data showed high genetic similarity among local and Asian populations. Both markers showed that <i>H. marginatum</i> and <i>D. marginatus</i> clustered with Turkish lineages. Molecular markers and haplotype analyses provide robust tools for accurate species identification, revealing tick diversity, population structure, and dispersal pathways. These insights are essential for effective surveillance and control of tick-borne diseases in the region.</p>