High electron mobility transistors (HEMTs) based on AlGaN/GaN heterostructures on silicon are stateof-the-art commercial power devices on the basis of nitrides. Performance and reliability of suchdevices are hampered by the presence of various defects. However, the relation between the natureof those defects and device characteristics is often not understood from a microstructural and physicalpoint of view. In this work we compare two nominally identical HEMT layer stacks that were grownunder different nucleation conditions, but differ significantly in vertical breakdown voltage. By defectselective etching it is shown that this phenomenon is not due to a different dislocation density. Insteada Conductive Atomic Force Microscopy study reveals locally strong electrical conductivity within thebuffer, which results in the formation of discrete vertical leakage paths that were identified tocorrespond to V-shaped defects at interfaces of the layer stack, also called V-pits. A detailed analysisby Cathodoluminescence (CL) mappings and CL-spectroscopy allows an identification of V-pits actingas centers of strongly reduced carbon and increased donor incorporation. Depending on growthconditions of the nucleation layer V-pits within the buffer occure in different sizes and densities acrossthe structure. The V-pits are closed by overgrowth sooner or later and are filled by the material of thefollowing layer. If such pits reach the upper buffer region, column-like areas of locally higher n-typebehavior are formed and disrupt the vertical device breakdown strength. The breakdown voltage isshown to be strongly dependent on the density of those V-pit induced n-type columns underneath theactive device region. An optimized nucleation layer growth was able to enhance the breakdownvoltage by nearly 600V.