Lepista nuda (Bull. ex Fr.) Cooke (Tricholomataceae) is an edible fungus with both economic and medical value. Identification of its mating-type loci is important for promoting breeding programs in L. nuda. The A mating-type locus of L. nuda and its flanking region were cloned and characterized in the present study. It contained two homeodomain transcription factor genes (called lna1 and lna2). Lna1 and Lna2 protein harbored conserved motif of homeodomain transcription factor protein. The novel finding of this study was that the gene order around the A locus was mip, lna2, lna1, and beta-fg in L. nuda, which was differed from other edible fungi. In addition, lna1 and lna2 showed divergent, inward transcriptional direction. The phylogenetic tree of HD proteins showed that L. nuda Lna1 and Lna2 were phylogenetically related with Laccaria bicolor. Our results revealed that the A mating-type locus had been subjected to gene rearrangements relative to all other basidiomycetes.