Abstract: Flammulina velutipes is an important commercial cultivation of edible fungi, with high nutritional and medicinal value. Based on the genomes of two monokaryotic strains, we analyzed the tRNAs in the two F. velutipes. In the 1 866 scaffolds of W23 genome, 204 tRNA genes were identified, including 25 possible pseudogenes, 3 unknown isoforms. In the 1 856 scaffolds of L11 genome, 194 tRNA genes were identified, including 27 possible pseudogenes, 5 unknown isoforms. The max tRNA length was 244 bp, and the min one was 58 bp. Total length was about 0.05% of the genome. There was no selenocysteine and possible suppressor tRNAs in both monokaryotic strains. Analysis of the tRNA coding genes will be helpful to further study the molecular function and phylogeny of F. velutipes evolution.