A genetic and physical map has been developed for the loculoascomycete Leptosphaeria maculans, a pathogen of oilseed Brassicas. The genetic map was constructed from 58 F1 progeny and comprises 155 amplified fragment length polymorphic (AFLP) markers, three random amplified polymorphic DNA (RAPD) markers, the mating type locus and a host specificity locus conferring the ability to form lesions on Brassica juncea. Twenty-one linkage groups, 5 pairs, and 18 unlinked markers were assigned, and the genome size was 1520 cM. Pulsed field gel electrophoresis experiments showed that the parental isolates each had 16 chromosomes and a genome size of about 33.5 Mb. Attempts to anchor a large number of markers to chromosomes were hampered by difficulties in converting AFLPs into RFLP markers, and because many markers bound to every chromosome, indicating that L. maculans has a high level of dispersed repetitive sequences. This fungus displays chromosomal length polymorphisms, but in the cross examined, the linkage and physical maps were essentially congruent and there was no evidence of translocations. The host specificity locus is 18 cM from the nearest AFLP marker and is located on a chromosome sized 1.85 Mb in the virulent parent. The mating type locus is on a chromosome sized 2.6 Mb and coincident on an AFLP marker amplified from the virulent parent. The derived amino acid sequence of part of this marker has some conserved amino acids present in the High Mobility Group DNA binding domain of MAT-2 mating type genes of other ascomycetes.