An electrophoretic karyotype of Aspergillus oryzae was obtained by transverse altering-field electrophoresis. Seven chromosomal bands were found. With Schizosaccharomyces pombe chromosomes as size standards, we estimated the sizes of the chromosomes to be 7.0, 5.2, 5.0, 4.5, 4.0, 3.7, and 2.8 megabase pairs (Mbp). The chromosomal DNA bands were identified with use of 13 cloned genes including ribosomal DNA. The intensity of ethidium staining and the results of Southern blotting with 100 random clones isolated from A. oryzae suggested that the smallest band migrated as doublet and that the total genome size was approximately 35 Mbp.