Transport of water, solutes, and contaminants through a thin film composite (TFC) membrane is governed by the intrinsic structure of its polyamide separation layer. In this work, we systematically characterized the nanoscale polyamide structure of four commercial TFC membranes to reveal the underlying structure-property relationship. For all the membranes, their polyamide layers have an intrinsic thickness in the range of 10-20 nm, which is an order of magnitude smaller than the more frequently reported apparent thickness of the roughness protuberances due to the ubiquitous presence of nanovoids within the rejection layers. Tracer filtration tests confirmed that these nanovoids are well connected to the pores in the substrates via the honeycomb-like opening of the backside of the polyamide layers such that the actual separation takes place at the frontside of the polyamide layer. Compared to SW30HR and BW30, loose membranes XLE and NF90 have thinner intrinsic thickness and greater effective filtration area (e.g., by the creation of secondary roughness features) for their polyamide layers, which correlates well to their significantly higher water permeability and lower salt rejection. With the aid of scanning electron microscopy, transmission electron microscopy, and tracer tests, the current study reveals the presence of nanosized defects in a polyamide film, which is possibly promoted by excessive interfacial degassing. The presence of such defects not only impairs the salt rejection but also has major implications for the removal of pathogens and micropollutants.