Two allelic variants of the gpd gene, Gpd(a) and Gpd(b), were isolated based on a putative glyceraldehyde-3-phosphate dehydrogenase encoding sequence from the transcriptome of Auricularia auricula-judae strain Au916. The two alleles were found to have a 73 bp length discrepancy and 39 SNP variations. Both of the genomic DNA sequences of two alleles were interrupted by five introns, and encoded a same 340 aa protein. Intron positions analysis showed that the first intron was absent, but the last unique intron was gained in A. auricula-judae. Allele-specific expression analysis showed that the Gpd(a) and Gpd(b) were expressed with no significant difference in dikaryotic mycelia of A. auricula-judae. To the best of our knowledge, this is the first report about the detection of two allelic gpd genes in A. auricula-judae, as well as the application of allele-specific primers in gene expression analysis for this edible fungus.