AbstractROOT HAIR DEFECTIVE SIX‐LIKE(RSL) genes control the development of structures from single cells at the surface of embryophytes (land plants) such as rhizoids and root hairs.RSLproteins constitute a subclass (VIIIc) of the basic helix–loop–helix (bHLH) classVIIItranscription factor family.The Charophyceae form the only class of streptophyte algae with tissue‐like structures and rhizoids. To determine if the function ofRSLgenes in the control of cell differentiation in embryophytes was inherited from a streptophyte algal ancestor, we identified the single classVIII bHLHgene from the charophyceaen algaChara braunii(CbbHLHVIII).CbbHLHVIIIis sister to theRSLproteins; they constitute a monophyletic group. Expression of CbbHLHVIIIdoes not compensate for loss ofRSLfunctions inMarchantia polymorphaorArabidopsis thaliana. InC. brauniiCbbHLHVIIIis expressed at sites of morphogenesis but not in rhizoids.This finding indicates thatC. brauniiclassVIIIprotein is functionally different from land plantRSLproteins. This result suggests that the function ofRSLproteins in cell differentiation at the plant surface evolved by neofunctionalisation in the land plants lineage after its divergence from its last common ancestor withC. braunii, at or before the colonisation of the land by embryophytes.