Nineteen single-conidiospore isolates of Pyrenophora tritici-repentis were isolated from leaf samples of wheat collected from Brazil, Germany, India, and Nepal, and one ascospore isolate was obtained from the United States for a virulence analysis study. Eight wheat cultivars were inoculated with a conidial suspension from each isolate, and percent necrotic leaf area was measured 5-6 days later using video image analysis. A highly significant interaction was found between cultivars of wheat and isolates of P. tritici-repentis. Cluster analysis of the data indicated only a moderate variation in virulence among the isolates of P. tritici-repentis tested. In a host resistance study, a significant interaction between 21 wheat lines and four geographically differing isolates also was detected. In a greenhouse study, 61 spring and 34 winter genotypes were evaluated for resistance to P. tritici-repentis. When 21 genotypes were reevaluated in a growth chamber with four isolates, 12 winter and six spring wheat cultivars and lines were resistant to moderately resistant.