Flammulina velutipes, known as winter mushroom in the family of Physalacriaceae, is the main edible and export mushroom with the third highest production after oyster and king oyster mushroom in Korea. However, as normal consumers regard F. velutipes as a simple subsidiary material, there is a limitation to increasing mushroom demand. In order to overcome the consumption limit and increase the differentiation of new varieties, it is necessary to breed varieties with enhanced functionality in consideration of consumer preferences. Therefore, the study was performed to analyze nutrient components and several useful functional substances with 26 genetic resources of F. velutipes. Analyses of inorganic compound(Ca, K, Mg) and 15 amino acids revealed that Strain 4148 had the highest content among the 26 strains. Beta-glucan, which increases immune activity and polyphenol, which exert antioxidant effects were higher in non-white strains than in white strains with a small number of exceptions. Among the five fatty acids, linoleic acid(an omega-6 fatty acid) and α-linolenic acid(an omega-3 fatty acid), were detected in six mushroom strains. α-linolenic acid, which was not found in five major mushrooms including oyster mushrooms, was identified in F. velutipes. The results of HPLC analysis showed that ‘Auram’ (Strain 4232) and ‘Baekseung’(Strain 4230) had the highest content of the stabilizing neurotransmitter GABA(15.38 μg/ml and 20.56 μg/ml, respectively) among non-white and white strains, respectively. Our findings provide useful information for breeding F. velutipes to obtain strains with enhanced functionality.