Barley is indeed a versatile cereal crop, valued for its uses in food, animal feed, and increasingly in biofuel production. As interest grows in developing new barley genotypes that are better adapted to diverse environmental conditions and production systems, integrating agro-morphological evaluations with molecular marker analyses in barley breeding programs is essential for developing new genotypes. It is necessary to explore the genetic diversity of those germplasm to predicate their responses to targeted environments and regions. The current study explored the phenotypic and genotypic relations among Saudi advanced germplasm to facilitate the development of superior barley cultivars suitable for desert environments. Molecular microsatellites (SSR) markers revealed considerable wide genetic variation among Saudi germplasm and checks. Population structure analyses revealed four main groups. Those groups were validated using similarity analyses and coefficients. As well, principal components analysis (PCA) and heat map analyses separated the studied genotypes into four main groups. The improved Saudi germplasm, selected from the barley breeding program, revealed considerably wide genetic and phenotypic diversities, indicating the feasibility of selection to improve for semi-arid conditions. The improved line KSU-BR-C/G-2 had the highest grain yield and harvest index in the first season. Rihana/Lignee was followed by the KSU-BR-C/G-2 genotype, with a grain yield averaging 6734.07 (kg ha−1), in the first season. KSU-BR-88-29-10 yielded 20,000 kg ha−1 for biomass yield. In the second year, KSU-BR-30-7 had the highest biomass yield, with 27,037.04 kg ha−1.