To establish a HPLC method to determine uridine and adenosine content of Ganoderma lucidum extract， and determine content of uridine and adenosine in Ganoderma lucidum extract in different habitats. Methods： The chromatographic column was Diamonsil C18 (2) (5 μ m， 250 × 4.6 mm)； the mobile phase acetonitrile was -0.04 M KH2PO4=9：91； the flow rate was 1 mL， min-1； the detection wavelength was 260 nm； the column temperature was 35 ℃. Results： The range of uridine and adenosine was respectively in 0.2~50 μ g·mL-1 and 0.1~20 μ g·mL-1， and the linear relationship between concentration and peak area were good； the average accuracy of uridine and adenosine were both between 99.91 to 102.12%； day and inter day RSD were less than 1.76%， and sample recovery rate were within 94.42% to100.50%. Among the extract of Ganoderma lucidum in the 6 different areas， the highest uridine content contained in Ganoderma lucidum in Fujian and highest adenosine content appeared in Jilin， and there were no statistical differences of uridine and adenosine content of Ganoderma lucidum in the other 5 places. Conclusion： The method is specific， accurate， reproducible， and can be used for determination of uridine and adenosine in ganoderma extract， which provides the reference and basis for evaluation of Ganoderma lucidum extract.