An RFLP map was constructed for Cochliobolus heterostrophus, the causal agent of southern corn leaf blight, to facilitate the cloning of pathogenicity genes. A total of 128 RFLP and 4 phenotypic markers were analyzed. Of these, 126 were linked to other markers and delimited 944 cM, or, at a maximum, 58% of the total map length. Several RFLPs were found tightly linked to Tox1, a pathogenicity gene. A previously hypothesized translocation tightly linked to Tox1 was also detected;The numbers and sizes of the chromosomes of the two strains crossed to make the RFLP map were investigated using pulsed field gel electrophoresis. Based on our best estimate, 15 A chromosomes and 1 B chromosome were in B30.A3.R.45 whereas 15 A chromosomes were in Hm540. The size range of the chromosomes was from about 1.3 Mb to about 3.7 Mb. The physical genome size was estimated to be 35-40 Mb. The average kb/cM ratio was calculated to be roughly 22-25, smaller than that of most organisms. The low kb/cM value should make the RFLP map a very powerful tool for cloning genes using various strategies;Both probes for making the RFLP map and cloned genes from C. heterostrophus were hybridized to the separated chromosomes to associate the linkage groups and cloned genes with individual chromosomes. The reciprocal translocation associated with Tox1 and other differences in chromosome arrangement between the parents were also verified by this method.