Rapid alkalinization factor (RALF) are small secreted peptide hormones that can induce rapid alkalinization in a medium. They act as signaling molecules in plants, playing a critical role in plant development and growth, especially in plant immunity. Although the function of RALF peptides has been comprehensively analyzed, the evolutionary mechanism of RALFs in symbiosis has not been studied. In this study, 41, 24, 17 and 12RALFswere identified inArabidopsis, soybean,LotusandMedicago, respectively. A comparative analysis including the molecular characteristics and conserved motifs suggested that the RALF pre-peptides in soybean represented a higher value of isoelectric point and more conservative motifs/residues composition than other species. All 94 RALFs were divided into two clades according to the phylogenetic analysis. Chromosome distribution and synteny analysis suggested that the expansion of theRALFgene family inArabidopsismainly depended on tandem duplication, while segment duplication played a dominant role in legume species. The expression levels of mostRALFsin soybean were significantly affected by the treatment of rhizobia. SevenGmRALFsare potentially involved in the release of rhizobia in the cortex cells. Overall, our research provides novel insights into the understanding of the role of theRALFgene family in nodule symbiosis.