iPBS retrotransposon markers, in other words, inter-primer binding site markers based on retrotransposon, have been helpful for the determination of genetic diversity in several plants. The study was evaluated using 10 iPBS molecular markers on the level of genetic diversity and genetic structure of alfalfa genotypes. A total of 280 alleles in 50 alfalfa genotypes (48 local genotypes and 2 commercial varieties) were obtained by 267 polymorphic markers with an average of 28 per locus, ranging from 9 to 41 alleles. The rate of polymorphism of the markers ranged from 86.36 to 100% with an average of 93.71%. The average number of polymorphic bands per marker was detected as 26.7. The mean PIC value and Dice's similarity index were calculated as 0.14 and 0.50 respectively. The results of UPGMA analysis, principal coordinate analysis (PCoA), and STRUCTURE demonstrated that the 50 alfalfa genotypes could be classified into 4 subpopulations, namely the Q1, Q2, Q3, and Q4. The Nei's genetic distances ranged from 0.0121 to 0.0359. iPBS markers and alfalfa genotypes used in this study may be used in studies of alfalfa breeding and germplasm conservation.