When no phenotypic screen is available, gene disruption in the koji-mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination. To achieve efficient gene disruption in the koji-mold, we developed a novel positive-negative selection method to enrich for homologous recombinants. The pyrG gene from A. sojae was used as a positive selection marker for transformants, and the oliC31 gene of A. nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated. In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A. sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method. Despite the fact that this gene is not transcribed in A. sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A. sojae.