The water exchange between the James River and the Elizabeth River, an estuary and sub-estuary system in the lower Chesapeake Bay, was investigated using a 3D numerical model. The conservative passive tracers were used to represent the dissolved substances (DS) discharged from the Elizabeth River. The approach enabled us to diagnose the underlying physical processes that control the expansion of the DS, which is representative of potential transport of harmful algae blooms, pollutants from the Elizabeth River to the James River without explicitly simulating biological processes. Model simulations with realistic forcings in 2005, together with a series of processoriented numerical experiments, were conducted to explore the correlations of the transport process and external forcing. Model results show that the upriver transport depends highly on the freshwater discharge on a seasonal scale and maximum upriver transport occurs in summer with a mean transport time ranging from 15–30 days. The southerly/easterly wind, low river discharge, and neap tidal condition all act to strengthen the upriver transport. On the other hand, the northerly/westerly wind, river pulse, water level pulse, and spring tidal condition act to inhibit the upriver transport. Tidal flushing plays an important role in transporting the DS during spring tide, which shortens the travel time in the lower James River. The multivariable regression analysis of volume mean subtidal DS concentration in the mesohaline portion of the James River indicates that DS concentration in the upriver area can be explained and well predicted by the physical forcings (r = 0.858, p = 0.00001).