In this study, we investigated the inter- and intra-genomic sequence variation of alpha-zein genes and their polypeptide expression in different maize genotypes, i.e. inbreds and a set of Lombardy open pollinated varieties, by analyzing their RFLP, coding nucleotides and 2-dimensional (2D) protein fractionation profiles. An extensive analysis of coding capacity of alpha-zein sequences in various genotypes and in the B73 reference inbred allowed us to assign 2D-spots to specific zein sequences. Moreover, we found that some genes reported to contain in frame stop codons are very likely expressed. Collectively these data allowed us to constitute two barcodes respectively based on nucleotide variation and on 2-D protein patterns that identify univocally each genotype