American lotus (Nelumbo lutea) is one of the two species in Nelumbo and has only yellow flower. Identification of total flavonoids showed wild American lotus contained almost only flavonols with quercetin 3-O-glucuronide to be the dominant pigment. The variation tendency of the total flavonol content was coincident with yellow color variation of petals during flower development. To understand the mechanism of accumulation and constituent of pigments in petals, three pivotal genes, NlFLS1, NlFLS2 and NlFLS3, which were predicted to encode flavonol synthases were isolated and characterized by analyses of basic bioinformatics, temporal and spatial expression patterns and enzymatic activity. Their temporal expression levels showed the same variation tendency, which was also consistent with the development-dependent variation of total flavonol content. Spatial expression patterns indicated the three genes should function in petals. All the three proteins were demonstrated to be bifunctional dioxygenase, possessing both flavonol synthase activity and flavanone 3-hydroxylase activity. Besides, other flavonol biosynthesis related genes were also investigated on their expression levels to give more clues on the mechanism. Substrate preferences of the three FLSs, substrate competitions between the FLSs and other flavonol biosynthesis related enzymes, and the greatly differential expression levels between F3’H (flavonoid 3’-hydroxylase) and F3’5’H (flavonoid 3’,5’-hydroxylase) contributed to the flavonol constituent in the petals of America lotus, namely abundant quercetin-derivatives while very few kaempferol-derivatives and myricetin-derivatives.