One of the greatest virus disease threats to wheat production in the Great Plains of the USA is Wheat streak mosaic virus (WSMV). Breeding programs have developed wheat varieties that are resistant or tolerant to WSMV infection, but these characteristics are climate dependent, and may also vary by WSMV isolate. We tested 10 spring and nine winter wheat (Triticum aestivum) varieties and two barley (Hordeum vulgare) varieties for resistance and tolerance to one WSMV isolate over four years. In spring wheat and barley, there were year by cultivar interactions in terms of resistance and tolerance. However, in winter wheat, yield losses due to WSMV were relatively consistent across years and varieties. Additionally, we tested the impacts of three WSMV isolates individually and in a mixture on twelve, two, and twelve varieties of spring wheat, barley, and winter wheat, respectively. Resistance and tolerance varied by isolate and cultivar, but there were no isolate by cultivar interactions. For spring wheat and barley, yield impacts were greater for two of the three single isolates than for the isolate mixture, whereas in winter wheat, the isolate mixture caused greater yield losses than the individual isolates. Overall, the results indicate that resistance and tolerance phenotypes were influenced by environmental conditions and by WSMV isolate or combination of isolates, suggesting that cultivar screening should be conducted over multiple years and with multiple virus isolates.