InGaN/GaN-based light-emitting diodes (LEDs) exhibit remarkable tolerance to high threading dislocation densities (TDDs), maintaining internal quantum efficiencies above 90% at room temperature even with TDD exceeding 108 cm−2. This robustness is attributed to V-shaped pits (V-pits), which form around threading dislocations (TDs) in InGaN/GaN quantum wells (QWs) and create potential barriers that inhibit carrier diffusion toward non-radiative dislocation cores. However, assessing the intrinsic non-radiative activity of dislocations in InGaN QWs is challenging, as standard metalorganic vapor-phase epitaxy-grown LED structures tend to exhibit V-pits. Those V-pits originate from the InGaN underlayer (UL) used to trap surface defects from high-temperature GaN growth, ensuring high QW efficiency. To investigate the genuine properties of TDs, we developed an InGaN UL that suppresses V-pits while maintaining surface defect-trapping capability. By adjusting growth conditions, we fabricated QWs with and without V-pits and compared their optical properties. Photoluminescence and cathodoluminescence measurements confirmed that TDs in InGaN QWs act as non-radiative centers. Time-resolved photoluminescence further revealed comparable capture cross sections for dislocations and point defects.