Abstract Three genetically marked, single–spore isolates of Septoria nodorum from wheat were passed through detached leaves of wheat cvs Blueboy and Coker 747 and the barley cv. Boone to produce three sub–isolates per original isolate. Each sub–isolate was cultured for three pycnidiospore generations on its respective host. Virulence of each sub–isolate on detached leaves of Blueboy, Caldwell, Coker 747, and NK81W701 wheat, and Boone and Surry barley was compared with that of the original single–spore isolate from which it was derived. In most cases, sub–isolates passed through wheat were significantly more virulent than the originals on wheat cultivars. They also were more virulent to barley than the original isolates but they were less virulent to barley than to wheat cultivars. Isolate × cultivar interactions were statistically significant (P < .0001) for isolates passed through wheat or barley and were greater than isolate × cultivar interactions among the original isolates. In seven of eight isolates passed through wheat or barley, only the original genetic marker was recovered after three generations, indicating that cross–contamination could not account for the observed change of virulence. In the single case of apparent contamination, of a sub–isolate, virulence declined. Copyright © 1990, Wiley Blackwell. All rights reserved