Objective: This study aimed to investigate the quality differences among different hybrid enoki mushrooms （Flammulina filiformis） strains and breed new strains with high quality. Methods: Two different protoplast monocytes of yellow enoki mushrooms Fv1923 were used as one of the parents to cross with 6 enoki mushrooms protoplasts （2 yellow and 4 white strains）, and a total of 12 yellow hybrid strains were obtained. Principal component analysis and cluster analysis were used to evaluate the free amino acids of 12 hybrid strains, and the flavor diversity of different protoplast hybrid strains was also evaluated. Results: The average contents of essential amino acids, non-essential amino acids and total amino acids were 7.71, 22.83 and 30.54 mg/g, respectively. Methionine was the first limiting amino acid of strains F1, F11, F15, F16, F21, F29, F39, F41 and F42. The amino acid components in 12 hybrid strains had a strong correlation, and the cumulative variance contribution rate of the four principal components was 87.15%, which better reflected the comprehensive information of amino acids in hybrid strains. The top two strains in comprehensive scores were hybrid strains of Shangyan 1820 and Fv1923. Cluster analysis showed that 12 strains were divided into 6 groups, which was consistent with the results of principal component analysis and reflected the variety differences of free amino acids in different strains. Conclusion: There were significant differences in the composition and content of free amino acids inhybrid strains with different protoplast monocytes as parents.