Abstract: Carotenoids are a kind of important natural pigments. In order to analyze the compositions and contents of carotenoids in pumpkin, pumpkin flesh was used as the material, different solvents (methanol, ethanol, petroleum ether, acetone, acetone: petroleum ether (1:1), acetone: petroleum ether (1:2) and acetone: petroleum ether (1:4)) and extraction methods (saponification and unsaponification) were screened to optimize the carotenoids extraction conditions in pumpkin flesh. Ultra Performance Liquid Chromatography (UPLC) technology was used to determine the carotenoid compositions and contents of 23 pumpkin cultivars. The results showed that the suitable extraction for carotenoids in pumpkin was ethanol solvent with saponification. Pumpkin flesh contained three carotenoid compositions, β - carotene, α- carotene and lutein, and the compositions and contents were both different in interspecies and intraspecies. Most of Cucurbita maxima and Cucurbita moschata cultivars had higher total carotenoid contents than Cucurbit pepo cultivars. The optimized extraction and determination methods in this study are suitable to the quality and quantity analysis of pumpkin flesh in China.