Cyclodextrin (CD) is known to afford excellent enantioselectivities due to its hydrophobic cavity and external H-bonding sites from hydroxyl moieties. However, there is still a lack of direct and comprehensive evidence clearly illustrating the origin of the important H-bonding effect. Regarding this issue, herein, four allylimidazole CD derivatives by selective substitution of the primary (6-position) and/or secondary (2,3-position) CD were synthesized and clicked onto silica surfaces to afford the corresponding chiral stationary phases (CSPs). The chiral chromatographic performances were systematically evaluated by separating 35 racemic analytes including isoxazolines, dansyl-amino acids, flavonoids and other racemates under reversed-phase HPLC. The chiral selection factors ( α ) and retention times ( k ) of the analytes on the as-prepared CSPs were comprehensively compared and it reveals that the enantioseparation ability was significantly altered due to the selective substituents of CD hydroxyl groups. The natural allylimidazole CD CSP (AICDCSP) was superior to the 6- O-tert -butyldimethylsilyl AICDCSP (6-TBDMAICDCSP) for most analytes. Dansyl amino acids and Ar-Pys were well separated on AICDCSP and 6-TBDMAICDCSP, where dansyl amino leucine gained the highest resolution up to 4.72 on AICDCSP, and flavonoids and Ar-Oprs were only separated on AICDCSP. These interesting separation results demonstrate that the secondary hydroxyl groups play a pivotal role in the separation of chiral compounds. In addition, the size of the CD cavity and the choice of solute also have an effect on the separation of substances. The mechanism involved in enantioselective discrimination of the selectively substituted CDs was further investigated by the molecular docking simulation.