Heat shock treatment of near isogenic barley lines induced susceptibility against powdery mildew (Blumeria graminis f. sp. hordei, Bgh). When barley lines were immersed into hot water (48–49 °C) for 20 seconds one day before inoculation with Bgh race A6, the heat treatment increased susceptibility in susceptible barley cv. Ingrid and in its near-isogenic barley lines carrying different effective resistance genes. Microscopic investigations indicated vigorous development of the pathogen not only on heat treated susceptible Ingrid and resistant Mla, but also on Mlg-resistant and even mlo-resistant lines. However, when longer heat stress was used, infection density increased gradually on the susceptible Ingrid leaves, and the 40–50 sec heat treatment induced the development of visible powdery mildew colonies even on mlo leaves. Heat stress significantly increased leakage of ions from leaf segments from all barley lines with or without specific resistance genes and caused a late decrease of SOD and a slight increase in CAT enzyme activities, which correlated with the slightly down-regulated levels of hydrogen peroxide in the heat treated barley leaves. Significant increase of RNase activities was found after heat stress, and there was a slight degradation of total DNA as a consequence of heat pretreatment in all barley lines.