Chemical and multivariate statistical analyses were used to evaluate how multiple sources of recharge and variations in aquifer flow paths influence the chemical composition of discharge at the River Rise, a first magnitude spring issuing from the eogenetic Upper Floridan Aquifer (UFA) in north-central Florida. Here, the upper Santa Fe River terminates at a swallet, called River Sink, where water enters the UFA and travels through a network of conduits for ~5 km before resurging at the River Rise. Chemical analysis of water from the River Sink, eight monitoring wells, four karst windows and the River Rise, shows water in the UFA reflects mixtures of three chemically-distinct sources including a calcium-bicarbonate type groundwater (Ca-HCO3), calcium-magnesium-sulfate type groundwater (Ca-Mg-SO4) and sodium-chloride type surface water (Na-Cl). Composition of River Rise discharge reflects variable mixing between these three end-members as rainfall and river stage affect flow to the spring from conduit and diffuse sources. During high river stage, surface water entering the River Sink dominates spring discharge; however, during low flow, Ca-HCO3 type groundwater, likely from the matrix porosity, drains into the conduits as head gradients reverse. Conversely, principle component analysis shows that 54% of the variance in this portion of the UFA results from the Ca-Mg-SO4 type water, which occurs at only one well. This well has the highest solute concentrations and an average temperature of ~5 °C above the other wells although all are about 30 m deep. These characteristics suggest deep water upwelling contributes to spring chemistry. We suggest that spatial variations in groundwater chemistry must be known to separate sources of water to springs, which cannot be resolved by only monitoring composition of spring discharge. Our results also demonstrate that water stored in matrix porosity can influence spring chemistry in eogenetic aquifers even where flow is dominated by conduits.