Groundwater discharge zones in streams are important habitats for aquatic organisms. The use of discharge zones for thermal refuge and spawning by fish and other biota renders them susceptible to potential focused discharge of groundwater contamination. Currently, there is a paucity of information about discharge zones as a potential exposure pathway of chemicals to stream ecosystems. Using thermal mapping technologies to locate groundwater discharges, shallow groundwater and surface water from three rivers in the Chesapeake Bay Watershed, USA were analyzed for phytoestrogens, pesticides and their degradates, steroid hormones, sterols and bisphenol A. A Bayesian censored regression model was used to compare groundwater and surface water chemical concentrations. The most frequently detected chemicals in both ground and surface water were the phytoestrogens genistein (79%) and formononetin (55%), the herbicides metolachlor (50%) and atrazine (74%), and the sterol cholesterol (88%). There was evidence suggesting groundwater discharge zones could be a unique exposure pathway of chemicals to surface water systems, in our case, metolachlor sulfonic acid (posterior mean concentration = 150 ng/L in groundwater and 4.6 ng/L in surface water). Our study also demonstrated heterogeneity of chemical concentration in groundwater discharge zones within a stream for the phytoestrogen formononetin, the herbicides metolachlor and atrazine, and cholesterol. Results support the hypothesis that discharge zones are an important source of exposure of phytoestrogens and herbicides to aquatic organisms. To manage critical resources within the Chesapeake Bay Watershed, more work is needed to characterize exposure in discharge zones more broadly across time and space.