Abstract Iran is remarkably rich in the genus Crataegus. Hawthorns can be used as a rootstock for pear and quince. The objective of the present study was to obtain an overview on the genetic relatedness among and within 201 accessions of genus Crataegus in Iran using three types of molecular markers. A total of 13 ISSR and 13 SCoT primers were used which produced 130 and 132 amplified fragments, of which 116 (88.94%) and 115 (86.54%) bands were polymorphic. Also, the total number of amplified alleles by 11 SSR primer pairs was 76 and the mean number of alleles per locus (Na) ranged from 3.81 to 5.44, while the mean number of effective alleles per locus (Ne) varied from 2.83 to 3.86. Genetic similarity (GS) by Jaccard’s similarity coefficient ranged 0.23–0.70 by ISSR, 0.02–0.62 by SCoT and 0.00–0.85 by SSR, indicating a high level of genetic variation that exists in the studied gene pool. The UPGMA dendrograms and Bayesian clustering analysis constructed based on all of the three molecular systems separated the genotypes into three main clusters with several sub-clusters indicating considerable genetic diversity in the studied germplasm. The information obtained here could be valuable for devising strategies for the germplasm conservation.