Triticum and Hordeum are two genera of Triticeae family with important cultivated species. Analysis of phylogenetic relationships of cultivated species and wild relatives of these genera is essential for their efficient use in breeding programs. In the present study, phylogenetic relationships of four Triticum and three Hordeum species were assessed using 150 barley microsatellite primer pairs. In total, 49 primer pairs amplified products in the both genera and 19 only in Hordeum species. Out of the 49 amplified markers, 21 were only polymorphic in Triticum species and in total, 59 alleles ranging from 2 to 5 with an average of 2.80 alleles per locus were amplified. In Hordeum genus using 43 polymorphic markers, 123 alleles with an average of 3.02 allele per locus and range of 2 to 14 were obtained. In two genera species, 18 polymorphic primer pairs amplified 91 alleles ranging from 2 to 19 with an average of 5.05 and gene diversity varied from 0.14 to 0.85 with a mean value of 0.59. Cluster analysis using Neighbor-Joining algorithm and Kimura-2-parameter distance coefficient and principal coordinate analysis separated the species of the two genera. In a cluster of Triticum genus, species were grouped based on their ploidy levels and bread wheat cultivars were separated from its wild relatives, and species with A genomes were grouped together. In Hordeum genus, cultivated barley was also separated from their wild relatives. Results of the present study show the efficiency of barley SSR markers in analysis of phylogenetic relationships of Triticum and Hordeum genera. In addition to reasonable transferability and polymorphism of the used markers in the species of both genera, they could also group the species based on their genomic constitution and evolutionary relationships.