A method for simultaneous separation and determination of four enantiomeric pairs of beta-blockers in waters by chiral CE has been developed. Off-line SPE was employed using functionalized ordered mesoporous silica as sorbent. Separation by CE was achieved using a BGE composed by methylated-beta-CD (1.25% w/v) dissolved in a 50 mM phosphate buffer (pH 2.5) and 30 degrees C, with good chiral resolution for all enantiomers. Mesoporous silica functionalized with octadecyl groups (denoted SBA15-C18) was prepared by a postsynthesis method and applied for the preconcentration of atenolol, propranolol, metoprolol, and pindolol enantiomers in waters by off-line SPE. Under optimized conditions, a preconcentration factor of 300 was achieved, employing 100 mg of SBA15-C18 as sorbent, with recoveries between 96 and 105% in tap water and good repeatability (% RSD = 711%, n = 6). Commercial C18 amorphous silica (ExtraBond(R) C-18) was also tested as sorbent for SPE, but results revealed better extraction capacity with higher recoveries for the SBA15-C18 material. The analytical characteristics of the off-line SPE-chiral CE method were evaluated, showing good precision, linearity, and accuracy with method quantification limits between 5.3 and 13.7 mu g/L for all enantiomers. The SBA15-C18 material allowed the extraction of four enantiomeric pairs of beta-blockers spiked in tap water, river water, and ground water with recoveries between 58 and 105%.