The newly developed Salixhybrids were assessed for genetic diversity using ISSR (Inter Simple Sequence Repeats) molecular markers. The results revealed 88.08 polymorphism, 0.53 polymorphism information content, 4.70 effective multiplex ratio, 2.54 marker index, and 7.18 resolving power from the amplified primers. Clones of the Salix matsudana(MAT) population recorded a maximum mean number of effective alleles (1.42), Shannon information index (0.38), gene diversity (0.25), and percentage of polymorphic loci (82.24 %). The AMOVA (Analysis of molecular variance) results revealed a moderate genetic divergence with a genetic variation of 88 per cent within populations and 12 per cent among the populations. The highest value of total diversity (0.28) and gene diversity (0.24) was recorded between S. matsudana(MAT) and S. tetrasperma(TETRA) populations, while there was more gene flow between S. matsudana(MAT) and S. tetrasperma(TETRA) populations (5.86). The clustering pattern obtained through STRUCTURE software showed two main gene pools and a mixture of 6–7 gene pools to confirm the hybrids based on ISSR marker data. S. matsudanaand S. tetraspermaclones showed the highest gene diversity due to their proximity from their original locations, resulting in the least amount of gene exchange among populations. Molecular characterization using ISSR markers was extremely useful for studying the genetic diversity among the willow hybrids and high genetic variability should be utilized for breeding purposes in the future.