This study aimed to identify and further characterize hydrophobin genes in the whole-genome sequence of Flammulina velutipes. Ten genes (Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10) sharing eight conserved cysteine residues were identified from 13 previously predicted putative hydrophobin genes in the F. velutipes genome. Quantitative real-time PCR (qPCR) analysis showed that these genes were specifically expressed in different developmental stages and tissues. Whereas all of the genes showed relatively higher levels of expression in the primordial stages of the fungus, most of them were expressed at relatively low levels in the mycelial stage.