Samples of bitter seeds of local ecotypes and cultivars of lupin (Lupin mutabilis), white and yellow ecotypes of quinoa (Chenopodium quinoa Wild) and a local ecotype of amaranth (Amaranthus caudatus) grown in the Peruvian highlands were analysed for total saponin content and sapogenol composition. Sweet cultivars of L albus and L luteus cultivated in mild-rainy lowlands of Chile were also analysed for comparison. Fast atom bombardment-mass spectrometry (FAB-MS) of the saponin extracts and gas chromatography (GC) analysis of the sapogenols after acid hydrolysis of the crude extract-were used for the identification and quantification of saponins. It was found that L albus and amaranth had undetectable levels of saponins making them attractive for human consumption. The cultivars and ecotypes of L mutabilis contained saponin levels in the range of 229.8–390.5 mg k−1. FAB-MS showed the presence of soya saponins I and II, whereas GC allowed the identification of soya sapogenols A and B. The same saponin composition was determined in L luteus whose total content was 55.3 mg kg−1. Saponin composition in quinoa seeds comprised oleanolic acid and three other sapogenols identified as hederagenin, phytolaccagenic acid and deoxyphytolaccagenic acid. Oleanolic acid saponins were found to be the main class of saponin in quinoa seeds sampled for this study. The yellow ecotype of quinoa presented a significantly higher content of saponins and of oleanolic acid as compared to white ecotypes. Since only one ecotype of amaranth was analysed, the nutritional significance of no detectable saponin needs further study. It was concluded that the environmental conditions in the Peruvian highlands are determinants of the amount and composition of saponins present in bitter lupine and quinoa.