AbstractRNA-induced silencing complex (RISC) plays a critical role in small interfering RNA (siRNA) and microRNAs (miRNA) pathways. Accumulating evidence has demonstrated that the major RISC members (AGO, DICER, TRBP, PACT and GW182) represent expression discrepancies or multiple orthologues/paralogues in different species. To elucidate their evolutionary characteristics, an integrated evolutionary analysis was performed. Here, animal and plant AGOs were divided into three classes (multifunctional AGOs, siRNA-associated AGOs and piRNA-associated AGOs for animal AGOs and multifunctional AGOs, siRNA-associated AGOs and complementary functioning AGOs for plant AGOs). Animal and plant DICERs were grouped into one class (multifunctional DICERs) and two classes (multifunctional DICERs and siRNA-associated DICERs), respectively. Protista/fungi AGOs or DICERs were specifically associated with the siRNA pathway. Additionally, TRBP/PACT/GW182 were identified only in animals, and all of them functioned in the miRNA pathway. Mammalian AGOs, animal DICERs and chordate TRBP/PACT were found to be monophyletic. A large number of gene duplications were identified in AGO and DICER groups. Taken together, we provide a comprehensive evolutionary analysis, describe a phylogenetic tree-based classification of the major RISC members and quantify their gene duplication events. These findings are potentially useful for classifying RISCs, optimizing species-specific RISCs and developing research model organisms.