Paleobotanical studies suggest that roots evolved at least twice independently during land plant diversification, once in lycophytes and once in euphyllophytes. Auxin promotes postembryonic root initiation in both groups but from different cell types. In several euphyllophytes, such as Arabidopsis, rice, and maize, AS2/LOB-domain (ASL/LBD) proteins act directly downstream of auxin and are conserved elements necessary for root initiation. It is currently unknown whether similar or different genetic mechanisms act downstream of auxin for root initiation in lycophytes and euphyllophytes. We searched for ASL/LBD proteins in genome sequences spanning the tree of life to retrace their evolutionary history. We performed a phylogenetic analysis of ASL/LBD proteins and mapped the functions of all characterized ASL/LBD onto the phylogenetic trees.We identified a clade specifically associated with root development, which includes no lycophyte sequence. This points toward the existence of distinct genetic mechanisms downstream of auxin for root initiation in lycophytes and euphyllophytes.