Lentinula edodes (shiitake/ Pyogo mushroom), the second most widely cultivated mushroom, is valued for foods and its pharmacological characteristics. In particular, recent reports have highlighted its medicinal properties, as well as the critical effects of its carbohydrates, including lentinan, on the immune. In addition to being an effective bio-reagent for degrading lignocellulosic wastes, L. edodes has also been shown to possess biotechnological utility. Genome sequencing and functional annotation will provide valuable information for establishing key molecular genetic markers that can be used to improve the quality and usage of this mushroom. In this project, we report whole genome de novo sequencing and gene annotation of L. edodes monokaryon B17. For whole genome sequencing, 61.6 X long reads (on average, 8,011 bp) were produced using the PacBio RS II system (Pacific Biosciences); 120 X long-mated pair reads (5 and 10 kb libraries) and 84.6 X short-insert reads (500 bp library) were produced using the Illumina HiSeq2500 system by 100 bp paired-end sequencing. The resulting assembly consists of 31 scaffolds constituting 46.1 Mb with 0.45% Ns (N50, 3.66 Mb; N90, 0.81 Mb; the longest scaffold, 5.85 Mb). Finally, 13,028 gene models were predicted by combining the results of transcriptome and protein sequence alignments and ab initio prediction, giving an average of 5.78 exons per gene.