Mushrooms(Pleurotus ostreatus, Agaricus bisporus and Flammulina velutipes) are popular food sources in Korea, and have been reported as therapeutic foods, useful for preventing various diseases. In this study we researched HPLC conditions for the determination of nucleic acids in extracts of the three type of mushrooms. The method for nucleic acids analysis of mushrooms was developed using HPLC with UV detection. To determine the nucleic acids, mushroom extracts were extracted in hot water at 90℃ by reflux extraction for 1 hr. Then, the extracts were hydrolyzed by enzymes RP-1G and 50000G. The HPLC conditions were simple, rapid, and sensitive, and were applicable for the analysis of 4 nucleosides (cytidine, uridine, guanosine and inosine) and 3 mono-nucleotides(5`-CMP, 5`-UMP, and 5`-IMP) in the mushrooms. The nucleic acids in the mushrooms were cytidine, guanosine, inosine, uridine, 5`-CMP, 5`-IMP, and 5`-UMP. The analysis results for total nucleic acids in the mushroom extracts(Pleurotus ostreatus, Agaricus bisporus, and Flammulina velutipes) indicated levels of 25.28, 27.75, and 19.87㎎/g, respectively. In conclusion, this method can be used successfully for qualitative and quantitative analysis of nucleic acids in Pleurotus ostreatus, Agaricus bisporus, and Flammulina velutipes.