is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96 %) of correct integration into the -locus of the genome of NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the -locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in To the best of our knowledge, this is the first report on the efficient site-specific integration at the -locus in the genome of .