The chain architecture dependence of the retention behavior of block copolymers in the temperature gradient interaction chromatography (TGIC) and liquid chromatography at the critical condition (LCCC) was investigated. For the purpose, polystyrene (PS)/polybutadiene (PB) diblock (SB), SBS triblock, and BSB triblock copolymers were prepared by sequential anionic polymerization and further fractionated by reversed-phase TGIC to obtain a set of the block copolymers with high purity, narrow distribution, and matched block length. In the TGIC separation with C18 bonded silica stationary phase and a mixture of CH2Cl2/CH3CN mobile phase, retention of the three block copolymers with matched molecular weight and composition shows a significant architecture effect: SBS elutes significantly earlier while BSB or SB elute later at similar retention volume. It indicates that the polymer-stationary phase interaction is less effective for the PB block located at the middle of the chain than the blocks located at the chain end. In LCCC separation at the critical condition for PB block, SBS is eluted early while SB and BSB were eluted later at the same retention time. Therefore, triblock copolymer with an invisible middle block behaves differently from those having invisible end block(s). This behavior is consistent with the theoretical prediction by Guttman et al.