Corn-leaf aphid (CLA), Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae) is a serious economic pest of barley worldwide. Breeding for aphid resistance in plants is considered a cost-effective and environmentally safe approach for aphid control, compared to the use of chemical pesticides. One of the challenges in breeding for aphid resistance is the identification of resistant plant genotypes, which can be achieved through the use of molecular markers. In the present study, a set of aphid specific 10 simple-sequence repeats (SSR) markers were used to investigate genetic diversity and population structure analyses in 109 barley genotypes against R. maidis. Three statistical methods viz., multivariate hierarchical clustering based on Jaccard's similarity coefficient, principal coordinate analysis (PCoA) and the Bayesian approach were utilized to classify the 109 barley genotypes. The analyses revealed four subpopulations i.e., SubPop1, SubPop2, SubPop3 and SubPop4 with 19, 46, 20 and 24 genotypes including admixtures, respectively and represented 17.43%, 42.2%, 18.34% and 22.01% genotypes of the total population size, respectively. The studied SSR markers produced 67 polymorphic bands, with an average of 6.7 and ranging from 3 to 12 bands. Heterozygosity (H) was found to be highest in SSR28 (0.64) and lowest in SSR27 (0.89). The observed genetic diversity index varied from 0.10 to 0.34 (with an average of 0.19). Major allele frequency varied from 74.08% to 94.80%. On an average, 87.52% of the 109 barley genotypes shared a common major allele at any locus. Based on the Aphid Infestation Index (AII), only 2 genotypes were found to be resistant against CLA. SubPop2 also had lowest mean aphid population (28.83), widest genetic similarity index (0.60-1.00) and highest genetic similarity coefficient (0.82), which highlighted its potential for inclusion in future CLA resistance breeding programs.