Zwitterionic, monodisperse-porous polymer beads were newly synthesized using a hydrophilic acrylic crosslinker (glycerol dimethacrylate, GDMA) or a hydrophobic one (ethylene dimethacrylate) including a zwitterionic functional monomer sulfopropyl-2-vinylpyridinium hydroxide (SVP) in a modified-staged shape template polymerization. The effects of monomer/seed latex ratio and diluent/seed latex ratio on the particle size, mean pore size, pore size distribution and specific surface area of the poly(1-(3-sulfopropyl)-2-vinylpyridinium hydroxide-co-glycerol dimethacrylate) poly(SVP-co-GDMA) and poly(1-(3-sulfopropyl)-2-vinylpyridinium hydroxide-co-ethylene glycerol dimethacrylate) poly(SVP-co-EDMA) microbeads were investigated. For both types of microbeads, the mean size decreased with decreasing monomer/seed latex ratio and decreasing diluent/seed latex ratio. Almost tenfold higher specific surface area was achieved using a hydrophobic crosslinking agent (EDMA) with respect to the hydrophilic one. Poly(SVP-co-GDMA) microbeads were satisfactorily used as a stationary phase in HILIC mode for the isocratic separation of polar organics with the plate heights up to 124 microns.