Dermacentor reticulatus, also known as ornate dog tick, is an important vector of the causative agents of various tick-borne diseases in humans, livestock and wild animals. The geographical range of D. reticulatus in Europe is fragmented and divided into two main zones: The Western European and the Eastern European zone. To investigate D. reticulatus population structure through its distribution range we used microsatellite markers and compared the genetic diversity of D. reticulatus in 26 selected sites in the western and eastern European distribution areas. A total of 254 unfed adult D. reticulatus ticks were collected from vegetation and genotyped at 5 microsatellite loci, and altogether 26 alleles were detected. The overall FST value of pairwise comparisons among 26 sampling sites was 0.128 and revealed a moderate genetic differentiation. Bayesian-clustering analysis showed that D. reticulatus forms two genetically distinct groups across Europe. Division of D. reticulatus distribution range into Eastern European and Western European populations with a possible recent overlap in Poland has been supported by molecular data obtained in this study.