In present study, a capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2′-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 μL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 °C, respectively. The optimized CE-MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138 ± 4823 μg/g for cultured C. sinensis; 3722 ± 1446 μg/g for C. militaris) than in natural ones (2167 ± 412 μg/g). However, the hypoxanthine (131 ± 47 μg/g) and inosine (335 ± 90 μg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5 ± 842.6 μg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.