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This letter presents a high isolation dual-frequency orthogonally polarized rectangular patch antenna utilizing microstrip feed line integrated with a defected ground structure (DGS). The demonstrated approach results in a significant improvement in port isolation in comparison to a conventional dual-polarized antenna fed by simple microstrip lines. Measurements show an improvement of 20 dB in port isolation relative to the conventional antenna, operating at 2 and 2.5 GHz. Image impedance of a microstrip line with DGS is controlled by the DGS geometry without modifying the dimension of the line. A 150 Ω high impedance line is effectively implemented using a microstrip line with 75 Ω line width by incorporating the DGS.