Mining throughout the Floridan aquifer system alters groundwater conditions. Determination of magnitude and extent of groundwater alterations in this regional karst aquifer system is difficult due to preferential flow paths, blasting, and widespread aquifer withdrawals and injections. Additionally, hydrologic models do not reveal subsurface impacts to the biotic environment, such as destruction of vegetative buffers, which are essential habitat for endangered and threatened species and other wildlife, and degradation from invasive species responding to hydroperiod disruptions. Our case study uses remote sensing and a geographic information system (GIS) to evaluate near-infrared (NIR) data for natural herbaceous depressional (wet prairie) wetlands in a central Florida phosphate mining area. These wetlands characteristically are dominated by graminoids, which have lower NIR reflectance and consequent digital numbers (DNs) in remotely sensed imagery than invasive species associated with hydroperiod alterations. Maximum aquifer withdrawals of 76,457 m3/d were permitted in November 1977 and remain active for study-area mines, which are surrounded primarily by un-irrigated pasture. Digital color infrared aerial imagery acquired in winter 2003–04 (1-m ground resolution) was used to extract NIR values within depressional wetlands. Digital historic (mid-1950s) U.S. Geological Survey topographic quadrangle maps (1:24,000 scale) were selected to delineate baseline wetland boundaries using ESRI ArcGIS 9.2. The study area included 567 of these wetlands, totaling 1,367 hectares. High mean NIR values (≥100 DN) consistent with hydroperiod-altered wetlands dominated by invasive plant species characterized 284 wetlands (50 and 60 percent total number and area, respectively), including some >5 km from study-area mines. Only 108 wetlands (20 and 10 percent total number and area, respectively) had low mean NIR values (≤80 DN) indicative of natural wet prairies without invasive species. Shallow ditches were not a statistically significant factor influencing NIR signatures in these wetlands. The spatial distribution of wetlands with high NIR DNs was inconsistent with conical groundwater drawdown predicted by groundwater models but suggests more linear fluid movement via subsurface preferential NW-SE flow paths.