OBJECTIVE： To establish a method for simultaneous determination of 5 nucleoside from different parts of cultured Cordyceps militaris. METHODS： HPLC method was adopted. The determination was performed on InertSustain C18 column with mobile phase consisting of methanol-0.02 mol/L monobasic potassium phosphate solution （gradient elution） at the flow rate of 0.6 mL/min. The detection wavelength was set at 254 nm， the column temperature was 30 ℃. RESULTS： The linear ranges of uridine， inosine， guanosine， adenosine and cordycepin were 0.568-3.408 μg（r＝0.999 9）， 0.284-1.704 μg（r＝0.999 9），0.264-1.584 μg（r＝0.999 9），0.232-1.392 μg（r＝0.999 9） and 1.672-10.032 μg（r＝0.999 8）， respectively. RSDs of precision， stability and repeatability tests were all lower than 3.0％. The recoveries were 98.2％-103.9％（RSD＝1.97％，n＝9）， 96.2％-101.6％（RSD＝1.76％，n＝9），96.7％-102.0％（RSD＝1.94％，n＝9），95.1％-99.4％（RSD＝1.43％，n＝9） and 95.6％-101.3％（RSD＝1.82％，n＝9）. CONCLUSIONS： The method is simple， precise， stable and repeatable， and can be used for simultaneous determination of 5 nucleoside from cultured C. militaris， the content of nucleosides are different in different parts of C. militaris.