The two-component signaling system-the major signaling pathway of bacteria-is found among higher eukaryotes only in plants, where it regulates diverse processes, such as the signaling of the phytohormone cytokinin. Cytokinin is perceived by a hybrid histidine (His) kinase receptor, and the signal is transduced by a multistep phosphorelay system of His phosphotransfer proteins and different classes of response regulators (RRs). To shed light on the origin and evolution of the two-component signaling system members in plants, we conducted a comprehensive domain-based phylogenetic study across the relevant kingdoms, including Charophyceae algae, the group of green algae giving rise to land plants. Surprisingly, we identified a subfamily of cytokinin receptors with members only from the early diverging land plants Marchantia polymorpha and Physcomitrella patens and then experimentally characterized two members of this subfamily. His phosphotransfer proteins of Charophyceae seemed to be more closely related to land plants than to other groups of green algae. Farther down the signaling pathway, the type-B RRs were found across all plant clades, but many members lack either the canonical Asp residue or the DNA binding domain. In contrast, the type-A RRs seemed to be limited to land plants. Finally, the analysis provided hints that one additional group of RRs, the type-C RRs, might be degenerated receptors and thus, of a different evolutionary origin than bona fide RRs.