Abstract A sensitive and reliable HPLC‐diode‐array detector method was developed for the first time to simultaneously determine nine nucleosides and nucleobases including uracil, cytidine, guanine, uridine, thymine, inosine, guanosine, thymidine and adenosine in 13 different Fritillaria species. The analysis was performed on a BaseLine C18 column with a gradient of acetonitrile in water at a flow rate of 0.8 mL/min. The diode‐array detector wavelength was set at 260 nm for the UV detection of nucleosides and nucleobases. Satisfactory separation of these compounds was obtained in less than 40 min. The optimized method provided good linear relation ( r 2 >0.9995 for all the investigated analytes), satisfactory precision (RSD <1.51%) and good recovery (from 97.64 to 101.16%). The established method was successfully applied to simultaneous determination of nine nucleosides and nucleobases in 61 batches of samples from 13 Fritillaria species collected from different habitats in China, which could be helpful to control the quality of Fritillaria bulbs.