This study investigated the genetic and environmental influences and their interactions on seed protein profiles of five narrow-leafed lupin cultivars grown under three different environmental conditions. High throughput MALDI-TOF mass spectrometry revealed 133 reproducible seed protein mass peaks. Thirty-one seed protein mass peaks were detected in all 15 combinations of cultivar × environment. Twenty mass peaks were influenced by cultivars irrespective of environment. Only six protein mass peaks were influenced by environments. Seventy-six mass peaks were highly variable. Number of mass peaks of lupin seed protein is mostly genetically controlled (P = 0.008) with no significant influence of the environment (P = 0.131). Environment and cultivar interactions were not significant (P = 0.889). Multivariate analyses of mass peak profiles supported the above analysis showing that protein mass peak profile was significantly (P = 0.001) influenced by cultivar but not by environment (P = 0.053). This result indicates the possibility of breeding new lupin cultivars targeting specific proteins for human food and animal feed without being too concerned about environmental influences.