The steps in the parasexual cycle required for genetic analysis by mitotic haploidization were readily accomplished in Aspergillus amstelodami. Balanced heterokaryons were synthesized between strains carrying complementary auxotrophic mutations and heterozygous-diploid colonies were recovered from these heterokaryons at frequencies between 1 × 10−6 and 5 × 10−6 among conidia plated. When grown in the presence of p-fluorophenylalanine or benomyl, heterozygous-diploid strains produced discrete haploid segregants. Examination of the segregation of 12 mutations among haploid segregants from ten diploids indicated that these genes were located in six linkage groups, thereby increasing the number of linkage groups recognized in this species. This haploidization analysis provided a clear distinction between linked and unlinked genes and continuation of this approach should reveal the chromosome number of A. amstelodami. Linkage at meiosis was detected between only one of five pairs of mitotically linked genes tested, emphasising the value of mitotic-haploidization analysis for assigning markers to linkage groups. A. amstelodami is amenable to both mitotic and meiotic genetic analysis and further studies should permit extensive comparisons of the genetic and biochemical organization of this species with that of A. nidulans.