Whole chromosomes from the fungal phytopathogen Leptosphaeria maculans were separated by transverse alternating field electrophoresis. The chromosome complements from several isolates of both highly and weakly virulent strains were compared. Small variations in chromosome size and apparent number were detected among isolates of the same strain. However, dramatic differences in both chromosome number and size were found when isolates of the highly virulen strain were compared to those of the weakly virulent. Highly virulent isolates had 6–8 distinct bands whereas weakly virulent isolates had 12–14. The genome sizes were estimated to be at least 8.6x106 base pairs for the virulent strain and 1.6x107 base pairs for the weakly virulent strain. The major differences found in the chromosome complements of the two strains, in combination with results of other biochemical, morphological, and genetic studies, indicate that they are distinct species.