Prevalence of tan spot of wheat caused by the fungus Pyrenophora tritici-repentis has become more prevalent in Oklahoma as no-till cultivation in wheat has increased. Hence, developing wheat varieties resistant to tan spot has been emphasized, and selecting pathogen isolates to screen for resistance to this disease is critical. Twelve isolates of P. tritici-repentis were used to inoculate 11 wheat cultivars in a greenhouse study in splitplot experiments. Virulence of isolates and cultivar resistance were measured in percent leaf area infection for all possible isolate x cultivar interactions. Isolates differed significantly (P < 0.01) in virulence on wheat cultivars, and cultivars differed significantly in disease reaction to isolates. Increased virulence of isolates detected increased variability in cultivar response (percent leaf area infection) (r = 0.56, P < 0.05) while increased susceptibility in cultivars detected increased variance in virulence of the isolates (r = 0.76, P < 0.01). A significant isolate × cultivar interaction indicated specificity between isolates and cultivars, however, cluster analysis indicated low to moderate physiological specialization. Similarity in wheat cultivars in response to pathogen isolates also was determined by cluster analysis. The use of diverse isolates of the fungus would facilitate evaluation of resistance in wheat cultivars to tan spot.