The Gibberella fujikuroi complex includes many plant pathogens of agricultural crops and trees, all of which have anamorphs assigned to the genus Fusarium. In this study, an interspecific hybrid cross between Gibberella circinata and Gibberella subglutinans was used to compile a genetic linkage map. A framework map was constructed using a total of 578 AFLP markers together with the mating type (MAT-1 and MAT-2) genes and the histone (H3) gene. Twelve major linkage groups were identified (n = 12). Fifty percent of the markers showed significant deviation from the expected 1:1 transmission ratio in a haploid F1 cross (P < 0.05). The transmission of the markers on the linkage map was biased towards alleles of the G. subglutinans parent, with an estimated 60% of the genome of F1 individuals contributed by this parent. This map will serve as a powerful tool to study the genetic architecture of interspecific differentiation and pathogenicity in the two parental genomes.