Pathogenic variability of 14 Septoria tritici isolates from different locations in the USA (California, Oregon, and Texas) was determined on seedlings of two sets of geographically diverse wheat cultivars under greenhouse conditions. Significant isolate effects, cultivar effects, and isolate × cultivar interactions were found, and a substantial amount of variation was accounted for by the interaction terms compared with the main effects of isolate and cultivar. All isolates were pathogenic on the cultivars tested but the degree of virulence on the individual cultivars varied among isolates. Linear contrasts between all homologous combinations (isolate × cultivar combination of same geographic location) and all heterologous combinations (isolate × cultivar combination of different locations) indicated that homologous combinations produced significantly more disease than heterologous combinations. The results demonstrate location-specific adaptation of S. tritici. Implications of pathogenic variability and local adaptation in S. tritici are discussed.