We evaluated the efficacy of using chemical characteristics to rank wetland relation to surface and groundwater along a hydrologic continuum ranging from groundwater recharge to groundwater discharge. We used 27 years (1974–2002) of water chemistry data from 15 prairie pothole wetlands and known hydrologic connections of these wetlands to groundwater to evaluate spatial and temporal patterns in chemical characteristics that correspond to the unique ecosystem functions each wetland performed. Due to the mineral content and the low permeability rate of glacial till and soils, salinity of wetland waters increased along a continuum of wetland relation to groundwater recharge, flow-through or discharge. Mean inter-annual specific conductance (a proxy for salinity) increased along this continuum from wetlands that recharge groundwater being fresh to wetlands that receive groundwater discharge being the most saline, and wetlands that both recharge and discharge to groundwater (i.e., groundwater flow-through wetlands) being of intermediate salinity. The primary axis from a principal component analysis revealed that specific conductance (and major ions affecting conductance) explained 71% of the variation in wetland chemistry over the 27 years of this investigation. We found that long-term averages from this axis were useful to identify a wetland’s long-term relation to surface and groundwater. Yearly or seasonal measurements of specific conductance can be less definitive because of highly dynamic inter- and intra-annual climate cycles that affect water volumes and the interaction of groundwater and geologic materials, and thereby influence the chemical composition of wetland waters. The influence of wetland relation to surface and groundwater on water chemistry has application in many scientific disciplines and is especially needed to improve ecological understanding in wetland investigations. We suggest ways that monitoring in situ wetland conditions could be linked with evolving remote sensing technology to improve our ability to better inform decisions affecting wetland sustainability and provide periodic inventories of wetland ecosystem services to document temporal trends in wetland function and how they respond to contemporary land-use change.