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Nucleic acid constituents are the main functional ingredients in edible fungi, therefore understanding the nucleic acid constituents of edible fungi often eaten on the table by the Chinese people is of significant value. In this study, Sichuan Provincial Center for Disease Control and Prevention collected 147 samples of edible fungi (including certain species that the Chinese often eat) from different parts of the Sichuan Basin. A new UPLC-QqQ/MS method has been developed to determine the 17 nucleic acid constituents in these 147 samples, including guanosine, adenosine, uridine, cytidine, inosine, thymidine, xanthosine dehydrate, 2′-deoxyguanosine, 2′-deoxyadenosine, 2′-deoxyuridine, 2′-deoxycytidine, 2′-deoxyinosine, guanosine 5′-monophosphate, adenosine 5′-monophosphate, uridine 5′-monophosphate, cytidine 5′-monophosphate, and inosine 5′-monophosphate. Finally, similarity assessment of the main edible fungus was performed using vectorial angle cosine method, and hierarchical cluster analysis was used to classify all the 147 samples. The results showed that some edible fungi have high similarities, especially in Lentinula edodes (Berk.) Pegler, the monogenic nucleotides content of which (55.84 ± 8.4 mg/100 g) is far greater than any other edible fungus, which is directly related to its taste. For quality control, this paper proposed to use the reference values of total nucleic acid compounds in edible fungi computed by percentile threshold method. This is the first time a comprehensive evaluation of nucleic acid constituents of different edible fungi of daily consumption was conducted for a large region, and the results is conducive to the quality evaluation and quality control of edible fungus. Unlabelled Image • Developed a new UPLC-/MS method for the determination of 17 nucleic acid constituents • The similarity was assessed by vectorial angle cosine and hierarchical cluster analysis. • The reference values of total nucleic acids in each species computed by percentile threshold method. • Can be applied for quality evaluation and quality control of edible fungus [ABSTRACT FROM AUTHOR]