OBJECTIVE： To set up a method for simultaneous determination of 5 nucleosides as uracil， hypoxanthine， uridine， creatinine and guanosine in Cervus nippon， to compare the differences of above nucleosides content in C. nippon among different producing areas. METHODS： HPLC method was adopted. The determination was performed on Aglient Eclipse Plus C18 column with methanol-0.07％ glacial acetic acid （4 ∶ 96，V/V） as mobile phase at the flow rate of 1.0 mL/min. The detection wavelength was 254 nm. The column temperature was set at 28 ℃， and sample size was 5 μL. Cluster analysis was conducted for 30 batches of C. nippon from different producing areas by using SPSS 19.0 software. RESULTS： The linear ranges of uracil， hypoxanthine， uridine， creatinine and guanosine were 0.001-0.01 mg/mL （all r≥0.999 9）. The limits of quantitation were 1.489 1， 1.927 9， 4.880 9， 7.884 6， 8.092 1 ng， and the limits of detection were 0.446 7， 0.578 4， 1.464 3， 2.365 4， 2.427 7 ng， respectively. RSDs of precision， stability， reproducibility tests were lower than 2％. The recoveries were 99.69％-103.65％（RSD＝1.40％，n＝9），97.77％-103.25％（RSD＝1.67％，n＝9）， 97.82％-101.81％（RSD＝1.12％， n＝9）， 99.30％-104.82％（RSD＝1.72％，n＝9） and 98.13％-100.20％（RSD＝0.64％，n＝9）. The contents of uracil， hypoxanthine， uridine， creatinine and guanosine were significantly different in 30 batches of C. nippon， which were clustered into 3 categories. S16-S18 and S22-S30 were clustered into one category； S8-S15 and S19-S21 were clustered into one category； S1-S7 were clustered into one category. CONCLUSIONS： The method has good precision， stability and reproducibility. It is suitable for simultaneous determination of uracil， hypoxanthine， uridine，  creatinine and guanosine in C. nippon. The quality of C. nippon has great difference due to ecological environment and technical factors of farming.