Lupins are an important source of protein that could replace soybeans in the diet of ruminants and monogastrics, without reducing their performance. Lupinus albus (L. albus) is the main species of the genus Lupinus that is cultivated in the Mediterranean region. The aim of the present research was to study commercial cultivars and advanced breeding lines of L. albus by using phenotypical, molecular and biochemical data, in order to be used in breeding projects. Seven commercial cultivars (Estoril, Fas Sweet, Multitalia, Magnus, Orus, Ulysse Sulimo and Figaro) and three advanced lines from the company AGROLAND (LKML, LKAP and LKAU) were used. Eleven morphological traits were described using UPOV Guidelines (International Union for the Protection of New Varieties of Plants). Additionally, agronomical traits and yield components were measured. Regarding the nutritional value, grain samples were analyzed for N and the crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), total alkaloids (TA), total phenolic content (TP), total tannins content (TT) and condensed tannins (CT) were calculated. Genetic diversity among genetic materials was assessed by SSRs molecular markers. The metabolomic analysis for four selected cultivars (Figaro, Magnus, Multitalia and Sulimo) was performed on the seeds with the GC/EI/MS technique. According to the results, the advanced lines were most productive but also with higher content of total alkaloids than the commercial cultivars. The only exception was the cultivar Multitalia that was characterized by a high content of alkaloids. Based on the SSRs, the cultivars Magnus, Orus and Estoril were grouped together while the breeding lines LKAP, LICML and LKAU were grouped with Multitalia. Regarding the metabolomic profile, the cultivars Multitalia and Magnus were together, while Sulimo was grouped with Figaro. Finally, the content of several beneficial metabolites for human and animal nutrition was significantly increased in Sulimo and Figaro, compared to Magnus and Multitalia. Both commercial varieties and lines have characteristics that can be exploited and used in breeding programs. Citation: Mavromatis, A.; Nianiou-Obeidat, I.; Polidoros, A.; Parissi, Z.; Tani, E.; Irakli, M.; Aliferis, K.A.; Zafeiriou, I.; Mylona, P.V.; Sarri, E.; et al. Characterization of Lupin Cultivars Based on Phenotypical, Molecular and Metabolomic Analyses. Agronomy 2023, 13, 370. https://doi.