Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), is an important pest of small grains cereals in many areas throughout the world. This research was conducted to determine the inheritance of resistance and to identify molecular markers of resistance in the barley (Hordeum vulgare L.) lines PI366444 and PI366453. Artificial infestation was performed in the field and growth chamber. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, Southern blotting, and polymerase chain reaction (PCR) techniques were used to determine plant genotypes. The PI366444 and PI366453 lines were intercrossed and each was also crossed with the susceptible cultivars Stark and Bearpaw. Allelism tests of F2 progeny showed that the resistant lines shared common or tightly linked resistance genes. Segregation ratios from crosses among resistant and susceptible plants, measured using F3 families, indicated that there were at least two resistance genes in both of the P1 lines. Two different regions in the barley genome were associated with RWA resistance genes. Variation for both leaf chlorosis and leaf rolling in F2 plants from crosses with both resistant lines was associated with the sequence‐tagged‐site (STS) PCR markers B‐hordein and D14 on the short arm of Chromosome 5. The STS‐PCR marker ABG8 on Chromosome 2 was associated with leaf rolling in one cross. Barley breeding programs throughout North America have devoted significant time and resources to backcrossing RWA resistance genes into acceptable cultivars. The molecular markers described in this report may assist barley breeding programs in introgression and fixation of linked resistance genes into useful germplasm.