Fifty lines of Medicago truncatula, derived from ecotypes or cultivars of diverse geographical origin, were grown under uniform conditions, and variation in seed protein composition and quantity was investigated. One-dimensional electrophoretic profiles revealed 46 major seed polypeptides, of which 26 were polymorphic within the collection. The vicilin/convicilin (7S) and the legumin (11S) type proteins were identified by immunoblotting using antibodies raised against the homologous pea proteins. The polymorphism for the major seed protein classes allowed the clustering of the genotypes into four groups. There was no evidence of clustering according to geographical origin of the lines. However, all lines not belonging to either M. truncatula ssp. truncatula or ssp. longispina were clustered in a single group, demonstrating the value of seed protein profiles in delimiting species boundaries. The Jemalong line group was differentiated early in the dendrogram, and thus represents an ancient clade in the seed diversity of M. truncatula. Within-accession variation was investigated for one-dimensional seed profiles, with additional lines obtained from the same ecotypes. As expected for an autogamous species, within-accession variation was low. Seed protein content was highly variable among the 50 lines examined. Lines contrasting for qualitative traits and seed protein content were identified to allow for the genetic determination of these characters.