Several studies have shown that Hordeum spontaneum contains potentially useful genes for improving agronomic traits of cultivated barley. In this study, the value of seven accessions of H. spontaneum C. Koch as donors of both cytoplasmic and nuclear genes affecting agronomic traits of barley were investigated;Favorable effects of H. spontaneum cytoplasm were found for straw yield, unit straw weight, and vegetative growth index. These favorable effects were actually attributed to nuclear-cytoplasmic interactions as indicated by the presence of cytoplasm-mating interactions and absence of cytoplasmic-generation interactions. Thus, H. spontaneum cytoplasmic genome must have interacted with H. vulgare nuclear genome. H. vulgare cytoplasmic effects were found to be superior to H. spontaneum for grain yield and harvest index. These cytoplasmic effects were attributed to nuclear-cytoplasmic interactions as indicated by the presence of cytoplasm-generation interactions. Therefore, H. vulgare cytoplasm must have interacted with either (a) H. spontaneum nuclear genes alone, or (b) specific combinations of H. spontaneum and H. vulgare nuclear genes;The seven accessions used in this study were assigned to four ecotypes and tested for variability among and within ecotypes by evaluating ecotype segregates. Superior performance for specific traits was, at least in part, a result of specific ecotypes. The most obvious effect was the superior straw yield performance from coastal ecotype segregates; however, these ecotype segregates also had the poorest harvest index. Segregates from matings involving northern and southern ecotypes had the highest harvest index. Based on the small sample size, variability among and within ecotype segregates suggests the possibility of obtaining superior progenies by sampling a larger number of accessions among and within ecotype categories. Results also further verify the utility of H. spontaneum ecotypes as donors of genes for agronomic traits in barley.