This study focuses on the structure-property relationship of interfacially polymerized thin-film composite (TFC) membranes. The impact of the polysulfone support layer on the polymerization of the polyamide active layer is investigated systematically and the resulting membrane surface morphology is related to RO separation performance under seawater desalination conditions. Six different TFC membranes with support layers having average pore sizes ranging from 18 nm to 120 nm were fabricated. Cross-flow RO tests showed that salt rejection systematically increased from 80.5% to 99.0% with decreasing support layer pore size. Scanning electron microscopy at high resolution revealed that the ridge-and-valley structure was more pronounced for active layers of TFC membranes prepared with support layers having larger pores. Convective monomer transport during interfacial polymerization is discussed as a possible reason behind the formation of ear- and ridge-like protuberances, of which the latter can apparently be damaging to the inner active layer.