Receptor mediated signal carriers play a critical role in regulation of plant defense and development. Rapid Alkalization Factor (RALF) is an important signaling family which has a role in plant growth and development. However, only few RALF polypeptides have been identified till date, mainly because of enormous efforts required for their isolation or identify their gene through mutational analysis. In this study, an extensive database search yield 39, 43, 34 and 23 potential RALF genes in Arabidopsis, rice, corn and soybeans, respectively. RALF genes are highly conserved across the plant species. A comprehensive analysis including the chromosomal location, gene structure, subcellular location, conserved motif, protein structure and promoter analysis was performed. RALF genes from four plants under study were divided in 7 groups based on phylogenetic analysis. In silico expression analysis of these genes, using microarray and EST data, reveled that these genes exhibit a variety of expression pattern. Furthermore, RALF genes showed distinct expression pattern under nitricoxide (NO) stress in Arabidopsis. This suggests a role of RALF genes in plant defense regulation. Our comprehensive analysis of RALF genes is a valuable resource that further elucidates the roles of RALF family members in plant growth and development. In addition, comparative genomics analyses deepen our understanding of the evolution of RALF gene family and will contribute to further genetics and genomics studies of other monocot and dicot plant species.