Electrophoretic karyotypes of ten strains of Mucor circinelloides f. lusitanicus were generated by contour-clamped homogeneous electric field (CHEF) gel-electrophoresis. Most of the strains analyzed showed polymorphisms, but a different main karyotype pattern could be correlated with each mating type. Genome structure was further analyzed by gene assignment to the chromosome-sized DNAs. Nonradioactive hybridization techniques identified the chromosomal localization of seven cloned genes. The hybridization patterns confirmed the similarity between the mating-type (−) strains and showed some heterogeneity among the mating-type (+) strains. Linkage was found between genes pyrF and chs3 in all the strains, between the gene leuA and the rDNA in all mating-type (−) strains and ATCC 1216b (+), and between chs2 and the rDNA in CBS 969.68 (+). This is the first time that gene linkage in M. circinelloides has been reported, but in some cases the linkage relationships obtained are strain-dependent.