Zwitterionic molecular brushes were generated on monodisperse-porous poly(3-chloro-2-hydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(HPMA-Cl-co-EDMA) beads (5.5 mu m in size) by surface initiated atom-transfer radical polymerization (SI-ATRP) of ([2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide) (MEDSPAH) via three different chemical routes. In the first route, triethanolamine was used as a spacer arm for attachment of surface initiator and SI-ATRP was initiated by chlorine moiety attached onto the spacer arm. In the second route, ethylenediamine was used as spacer arm and SI-ATRP was initiated with bromine moiety attached onto the spacer arm. In the third one, SI-ATRP was directly initiated with the chlorine moiety of HPMA-Cl units of the beads without using a spacer arm. Poly(MEDSPAH) brushes with the highest surface concentration and the shortest chain length were generated on the polymer beads by an SI-ATRP process initiated with bromine attached onto the surface with the spacer arm. The formation of higher amount of molecular brush with the shorter chain length was explained by higher radical generation rate of bromine as the surface initiator. Poly(MEDSPAH)-grafted poly(HPMA-Cl-co-EDMA) beads produced with different chemical routes were utilized as stationary phase for the separation of polar analytes in HILIC mode. Better chromatographic separations were obtained with the stationary phase carrying zwitterionic molecular brushes with the longest chain length. Chain length of the zwitterionic ligand was also more effective with respect to its surface concentration for better separation of nucleosides, nucleotides, acrylic compounds, and organic acids in HILIC mode.