Hawthorn (Crataegus spp.) is an edible wild fruit that is used in traditional medicine, landscape studies, and food and beverage industries in many countries. It is an important wild plant species in Turkey and is numerous in the Yozgat Province. Genetic and breeding studies on hawthorn are very limited. Therefore, we aimed to characterize 91 hawthorn genotypes using simple sequence repeat (SSR) markers. The SSRs were developed from apple and pear and were screened in hawthorn for amplification and polymorphisms. A total of 265 alleles were detected from thirty-two SSR primer pairs, and those were used to identify genetic relationships. The number of alleles ranged from 2 to 21 alleles per locus with a mean value of 8.28. The Hi05b09 locus showed the highest allele number (Na = 21). The polymorphism information content (PIC) values ranged from 0.16 (CH03d10) to 0.89 (C6554) with a mean value of 0.60. An Unweighted Pair Group Method with Arithmetic Average method was used to cluster the genotypes, and four major clusters were obtained from the amplification of the SSRs. STRUCTURE software identified four populations (Delta K = 4) and eight sub-populations (Delta K = 8), and four major clusters similar results to UPGMA analysis. Our study showed that the SSR markers could be utilized as a reliable tool for the determination of genetic variations and relationships of hawthorn genotypes. A basic molecular analysis on the hawthorn genotypes identified in this study will promote the collection of germplasm collection and the selection of parents' in future cross-breeding studies.