Studies on phylogeography and population structure of Ixodes ricinus have been carried out in Europe for decades, but the number of specimens from the Middle East included in these analyses is relatively small, despite the wide distribution of the species in this area. This study aimed to clarify the phylogenetic positions of I. ricinus from Turkey as well as to investigate the presence of Ixodes inopinatus in Anatolia. For this purpose, one mitochondrial (mt 16S rDNA) and one nuclear gene (defensin) were used to generate molecular data from I. ricinus samples, which were collected from 17 locations across the species' distributional range in Turkey. Bayesian inference was used to investigate phylogenetic relationships. Globally, the mt 16S rDNA lineages correspond to the lineages revealed by defensin; I. ricinus and I. inopinatus sequences clustered separately. However, a discordant genetic pattern was observed between the phylogenetic position of turkish I. ricinus revealed by nuclear versus mitochondrial genes. All Turkish haplotypes of mt 16SrDNA clustered with I. ricinus samples from Europe, which might be the result of extensive gene flow between populations of Europe and the Middle East. On the other hand, a sample from Thrace Region grouped within I. inopinatus clade. Thus, the occurrence of I. inopinatus in Turkey was demonstrated for the first time using molecular data. Moreover, four individuals were found to be heterozygous for the defensin. The potential evolutionary processes that underlie this observed discrepancy between the phylogenetic trees of two genes have been discussed.