The role of threading dislocations in the intrinsic degradation of lateral GaN devices during high reverse bias stress tests (RBSTs) is largely unknown. We now present the results on lateral p-GaN/AlGaN/2DEG heterojunctions with a width of 200 μm in GaN-on-Si. A time-dependent permanent degradation of the heterojunction under high reverse bias and elevated temperatures can be observed, ultimately leading to a hard breakdown and device destruction. By using an integrated series p-GaN resistor, the device is protected from destruction and, consequently, the influence of dislocations on the degradation mechanism could be studied. Localization by emission microscopy could show that the transient current increase during a RBST is the result of the creation of a limited amount of highly localized leakage paths along the whole device width. We could establish a 1:1 correlation of leakage sites with a structural material degradation within the AlGaN barrier for nine individual positions on two different devices by planar transmission electron microscopy analysis. To unambiguously show whether dislocations in GaN-on-Si even should be considered a potential trigger for the RBST degradation in lateral heterojunctions, a combined planar and cross-sectional lamella approach was used for the first time for larger devices. This enabled the visualization of the three-dimensional propagation path of the dislocations close to the degradation sites. It was found that there is no statistically significant link between the material degradation and pre-existing dislocations. Our findings offer new insights into the GaN-on-Si material system, upon which upcoming power technologies are built upon.