Abstract: This paper presents a substrate-dependent performance evaluation of a defected ground structure (DGS)-integrated multiband hexagonal microstrip patch antenna operating in the 22–28 GHz millimetre-wave band for 5G FR2 applications. To examine the influence of dielectric properties on electromagnetic behaviour, the same antenna geometry is implemented on three commonly used substrates—Duroid (relative permittivity εr ≈ 2.2, loss tangent tanδ ≈ 0.0009), Rogers (εr ≈ 2.94, tanδ ≈ 0.0012), and FR4 (εr ≈ 4.4, tanδ ≈ 0.02). A controlled substrate-based comparison is conducted with respect to the reflection coefficient, impedance bandwidth, gain, and radiation efficiency. The results indicate that substrate characteristics significantly affect resonance depth, impedance stability, and radiation performance at millimetre-wave frequencies. The Duroid-based configuration achieves S₁₁ below −32 dB, peak gain of 8–8.5 dBi, and high radiation efficiency due to reduced dielectric loss. The Rogers substrate exhibits stable multiband behaviour with moderate gain, whereas the FR4-based design shows reduced resonance depth and lower gain due to increased dielectric dissipation. By maintaining identical geometry across all substrates, the study isolates the direct impact of dielectric constant and loss tangent on modal excitation and efficiency degradation in the 22–28 GHz band. The presented analysis supports informed substrate selection for compact multiband mmWave antenna designs in next-generation wireless systems.