Hyalomma marginatum is a hard tick species of medical and veterinary importance that is widely distributed throughout Turkey. In this study, five novel polymorphic microsatellite markers were isolated and developed from partial genome information using the Hiseq Illumina paired end technology. A total of 20,943,321 reads were obtained from vegetation collected specimens, 103 primer pairs were randomly selected for the analysis and 62 of them were tested for polymorphism. Seventy five specimens from nine different localities of Turkey were genotyped. Markers for selected loci were tested using labeled primers on capillary electrophoresis. Five loci were successfully scored and used to analyze genetic diversity. The number of alleles per locus ranged from 4 to 7, with an average of 5.4 alleles per locus. The average observed and expected heterozygosity values were 0.314 and 0.468, respectively. Two loci deviated significantly from Hardy-Weinberg expectations. No significant linkage disequilibrium was detected between loci. This study is the first attempt to generate genome sequence data and to develop SSR markers for H. marginatum. These markers will be useful to understand the taxonomic status of this vector, its population dynamics, and spatial distributions as well as mechanisms underlying Crimean Congo Heamorrhagic Fever transmission and the development and implementation of well-designed prevention strategies.