Nodules cystine-rich (NCR) peptides from legume nodules mediate bacteroid differentiation of Rhizobium sp. This study aimed to characterize NCR peptides of Phaseolus vulgaris, Trifolium repens, Medicago sativa, and Vicia faba, as relevant information on these legumes is still lacking. After exposing the root nodules of four leguminous plants to sonication, the peptides were separated according to their molecular weight by sodium dodecyl gel electrophoresis (SDS-PAGE). The NCR peptide was detected by high-performance liquid chromatography (HPLC), followed by the determination of the protein sequence and amino acid composition and their evolutionary history in terms of the phylogenetic tree by bioinformatics analysis. The results showed these four peptides had the same molecular weight of 60 kDa. The peaks of the curve varied with retention time depending on the legume from which the peptide was extracted. Bioinformatic analysis of the results showed that the amino acid substitutions varied in number and sequence for each species and that these peptides had three distinct wire, ribbon, and surface patterns within the structural modeling of their crystals. Side chains of amino acids in the loops and helices within the grid box may facilitate hydrophobic interactions via tyrosine, phenylalanine, or polar interaction sites via cysteine, histidine, and glutamine. These loops often serve as flexible regions to adapt to binding partners. In general, this study contributed to the characterization of NCR peptides that showed mismatches and differences in amino acid sequences and deletions of some amino acids at different locations depending on the type of legume from which the peptide was extracted.