A capillary zone electrophoresis method for the simultaneous determination of twenty polyphenols in wine was developed. The separation was performed using fused-silica capillaries of 75 µm I.D. and a 30 mM sodium tretraborate buffer solution at pH 9.2 with 5% isopropanol as a background electrolyte. A capillary voltage of +25 kV with pressure-assisted (3.5 kPa) separation from min 18 was applied, thus, achieving a total analysis time lower than 20 min. Instrumental quality parameters such as limits of detection (LOD values between 0.3 and 2.6 mg/L), linearity (r2>0.990), and run-to-run and day-to-day precisions (RSD values lower than 6.5% and 15.7%, respectively) were established. Three different calibration procedures were evaluated for polyphenol quantitation in wines: external calibration using standards prepared in Milli-Q water, standard addition, and pseudo-matrix matched calibration using wine as a matrix. For a 95% confidence level, no statistical differences were observed, in general, between the three calibration methods (p-values between 0.11 and 0.84), while for some specific polyphenols, such as cinnamic acid, syringic acid and gallic acid, results were not comparablewhen external calibration used. CZE method using pseudo-matrix matched calibration was then proposed and applied to the analysis of polyphenols in 49 Spanish wines, showing satisfactory results and a wide compositional variation between wines. Electrophoretic profiles and other compositional data (e.g., peak areas of selected peaks) were considered as fingerprints of wines to be used for characterization and classification purposes. The corresponding data were analyzed by PCA in order to extract information on the most significant features contributing to wine discrimination according to their origins. Results showed that a reasonable distribution of wines depending on the elaboration areas was found, being tirosol, gallic, protocatechuic, p-coumaric and caffeic acids some representative discriminant compounds.