The SSR markers expressed a high level of genetic diversity (H_O = 0.438, and H_E = 0.437). Genetic differentiation among the populations was moderate (F_ST = 0.124), and genetic variation within the populations (79%) significantly surpassed among populations (21%). The gene flow (N_m = 1.840) may have remarkably influenced the population genetic structure of J. regia, which can be attributed to anthropological activities and wind dispersal of pollen. The STRUCTURE analysis divided the 27 populations into two main clusters. Comparing the neighbor-joining and principal coordinate analysis dendrograms and the Bayesian STRUCTURE analysis revealed the general agreement between the population subdivisions and the genetic relationships among the populations. However, a few geographically close populations dispersed into different clusters. Further, the low genetic diversity of the Sulaymaniyah (SMR) population of Iraq necessitates urgent conservation by propagation and seedling management or tissue culture methods; additionally, we recommend the indispensable preservation of the Gonabad (RGR) and Arak (AKR) populations in Iran.

These results reflected consistent high geographical affinity of the accession across the plateau. Our findings suggest that gene flow is a driving factor influencing the genetic structure of J. regia populations, whereas ecological and geological variables did not act as strong barriers. Moreover, the data reported herein provide new insights into the population structure of J. regia germplasm, which will help conserve genetic resources for the future, hence improving walnut breeding programs’ efficiency.