Bamboo shoots are regarded as potential sources of sterols. The effects of genetic variability, parts and harvest seasons on the sterol content and composition in the bamboo shoots have been determined using a novel ultra-performance liquid chromatographic atmospheric pressure chemical ionisation mass spectrometer method. The results showed that the representative sterols in bamboo shoots were β-sitosterol, campesterol, stigmasterol, ergosterol, cholesterol and stigmastanol; exception stigmastanol, the significant differences were observed in the sterol content of different species (112.4–279.6mg/100g dry wt), different harvest seasons (195.3–279.6mg/100g dry wt) and different parts (253.6–321.8mg/100g dry wt); the sterol composition was similar in different species and different harvest seasons, however, it was significantly different between shoot bodies and shoot shell. The genetic variability, parts and harvest seasons could significantly affect the sterol composition in the bamboo shoots. The spring shoot shell of Phyllostachys pubescens contained the highest sterol content (321.8mg/100g dry wt).