Substrate‐binding proteins (SBPs) mediate ligand translocation and have been classified into seven clusters (A‐G). Although the substrate specificities of these clusters are known to some extent, their ligand‐binding mechanism(s) remain(s) incompletely understood. In this study, the list of SBPs belonging to different clusters was updated (764 SBPs) compared to the previously reported study (504 SBPs). Furthermore, a new cluster referred to as cluster H was identified. Results reveal that SBPs follow different ligand‐binding mechanisms. Intriguingly, the majority of the SBPs follow the ‘one domain movement’ rather than the well‐known ‘Venus Fly‐trap’ mechanism. Moreover, SBPs of a few clusters display subdomain conformational movement rather than the complete movement of the N‐ and C‐terminal domains.