Uniformly sized functionalized macroporous polymer beads were prepared by either a classical copolymerization method or recently reported in situ surface modification method utilizing chiral methacrylamide as a functional modifier. To evaluate conformational and/or specific differences in their surface chiral functionality, we applied chromatographic evaluation techniques. The prepared modified beads were utilized as chiral stationary phase in high-performance liquid chromatography (HPLC). Those prepared by the in situ surface modification method tended to show higher chiral recognition ability than those by the classical copolymerization method, even if the equivalent amount of the chiral functional group was involved within polymer beads. Detailed chromatographic studies exhibited the in situ surface modification method could lead to polymeric methacrylamide functionality on the surface within relatively large pore size regions of the macroporous polymer beads, while the classical copolymerization method tended to form less polymeric surface functionality. The difference in the chiral surface functionality on both of macroporous polymer beads afforded drastic change in chromatographic chiral recognition ability. Complete resolution of a drug, thalidomide could be achieved on the chiral stationary phase with the polymeric chiral surface functionality, while no resolution was found on that with the monomeric one even if the same chiral methacrylamide was used as a modifier to prepare the chiral stationary phases.