Understanding the transport resistance of water molecules in polyamide (PA) reverse osmosis (RO) membranes at the molecular level is of great importance in guiding the design, preparation, and applications of these membranes. In this work, we use molecular simulation to calculate the total transport resistance by dividing it into two contributions: the interior part and the interfacial part. The interior resistance is dependent on the thickness of the PA layer, while the interfacial resistance is not. Simulation based on the 5 nm PA layer reveals that interfacial resistance is the dominating contribution (> 62%) to the total resistance. However, for real-world RO membranes with a 200 nm PA layer, interfacial resistance plays a minor role, with a contribution below 10%. This implies that there is a risk of inaccuracy when using the typical method to estimate the transport resistance of RO membranes, as this method involves simply multiplying the total transport resistance of the simulated value based on a membrane with a 5 nm PA layer. Furthermore, both the interfacial resistance and the interior resistance are dependent on the chemistry of the PA layer. Our simulation reveals that decreasing the number of residual carboxyl groups in the PA layer leads to decreased interior resistance; therefore, the water permeability can be improved at no cost of ion rejection, which is in excellent agreement with the experimental results.