Four Mexican Nymphaea species, N. ampla, N. pulchella, N. gracilis and N. elegans belonging to subgenera Brachyceras were analyzed. In this work two 5-glycosyl isoflavones, 7,3',4'-trihydroxy-5-O-beta-D-(2''-acetyl)-xylopyranosylisoflavone (1) and 7,3',4'-trihydroxy-5-O-alpha-L-rhamnopyranosylisoflavone (2), were isolated from N. ampla and N. pulchella, respectively, together with other known 3-glycosyl flavones and triterpene saponins from the same four species. The structures were elucidated by 1D and 2D NMR, FABMS, and other spectroscopic analyses. These results confirmed that the four species were different from each other and established that N. pulchella represents a different taxa than N. ampla. In addition, the 5-glycosyl isoflavones could be considered as a taxonomic character of this group of plants.