Cordyceps sinensis is a well-known traditional Chinese medicine, and some of the active components are nucleosides. The analysis of nucleosides in Cordyceps material has been performed by reversed-phase high-performance liquid chromatography (HPLC) with gradient elution or by spectrometry. Here, we have explored the possibility of using capillary electrophoresis to determine the content of three major nucleosides (adenosine, guanosine and uridine) in Cordyceps. Capillary electrophoresis needs no gradients, and it provides a better separation due to its higher efficiency. In order to optimize the resolution, the separation of adenosine, guanosine and uridine was determined in Cordyceps with respect to the variation of buffer concentration, pH, temperature, and voltage. By using the calibrated electrophoresis system, the separation was achieved for the three nucleosides in less than 10 min with a background electrolyte consisting of 0.2 M boric acid-sodium hydroxide buffer, pH 8.5. The nucleoside contents of various types of natural Cordyceps and cultured Cordyceps mycelia were determined and compared. There was a great variation of nucleoside content in different sources of Cordyceps; the cultured Cordyceps mycelia, however, contains a much higher concentration than the natural Cordyceps.