The present study supports the view that the retinoblastoma functions are shared by two distinct retinoblastoma-related (RBR) protein subfamilies in the monocot cereal species, whereas dicot plants have only a single RBR protein. Genes encoding RBR proteins were identified and characterized in alfalfa (Medicago sativa), rice (Oryza sativa), and wheat (Triticum aestivum). The alfalfa MsRBR gene encodes a new member of the dicot RBR proteins (subfamily A). A comparison was made of two rice genes, OsRBR1 (subfamily B) and OsRBR2 (subfamily C), which exhibit differences in exon-intron organization and share only 52% amino acid sequence identity. The plant RBR proteins can be categorized into three distinct subfamilies, in which the similarity between members is greater than the similarity to other RBR proteins from the same species. Comparison of the transcript levels in various tissues revealed that the expression of the OsRBR1 gene was high in embryos or cultured cells and gradually decreased from the basal region to the tip of the leaves. The OsRBR2 gene displayed more transcripts in differentiated tissues, such as leaves and roots. In contrast, the mRNA level of the MsRBR gene did not differ significantly in either mature leaves or cultured cells. The results of yeast two-hybrid pairwise interaction assays demonstrated differences between the rice RBR variants in the interactions with the phosphatase 2A B'' regulatory subunit and an unknown protein. The in silico and functional data presented in this work highlight considerable differences between dicot and monocot species in the retinoblastoma regulatory pathways and permit an improved classification of RBR proteins in higher plants.