We aimed to study the genetic diversity, population structure, and phylogeny of Iranian orchids using inter-simple sequence repeat (ISSR) markers to find markers associated with phenotypic traits. Based on the phenotypic analysis, the inflorescence length and the flower number of studied accessions ranged from 3.92 to 27.13 cm and 5 to 50, respectively. On the other hand, the tuber length ranged from 1.80 to 9.35 cm. A total of 310 reproducible ISSR fragments with a size range of 150 to 3000 bp were amplified. ISSR primers provided an average polymorphism information content of 0.391, varied from 0.488 for UBC-876 to 0.351 for UBC-842. Os.J population showed the lowest genetic diversity (H = 0.057 and I = 0.075), while Oyst.JA population showed the highest genetic diversity (H = 0.114 and I = 0.158). At species level, the average coefficient of genetic differentiation (GST) ranged from 0.265 for Orchis simia to 0.587 for Himantoglossum affine. Gene flow (Nm) varied from 1.38 (O. simia) to 0.756 (Anacamptis collina). The UPGMA genetic similarity dendrogram using Jaccard coefficients (r = 0.973) revealed six main clusters. Based on the Bayesian clustering method, the highest probability of the data was achieved when accessions were divided into eight groups. Floral and tuber-related phenotypic traits represented high correlations together, and they were associated with some ISSR bands based on the multiple association analysis. Altogether, ISSR markers proved to be useful for discrimination and clarification of the relationships among species and populations collected from geographically different locations. Furthermore, it could identify the polymorphism among accessions within each population and species.
