Lake eutrophication is a problem in many areas of Ontario, although the history of nutrient enrichment is poorly documented. The aim of this study was to construct a diatom-based transfer function to infer past phosphorus levels in Ontario lakes using paleolimnological analyses. The relationship between diatom assemblages and limnological conditions was explored from a survey of diatoms preserved in the surface sediments of 64 Southern Ontario lakes, spanning a total phosphorus gradient of 0.004 to 0.054 mg L-1. Over 420 diatom taxa were identified, 98 of which were sufficiently common to be considered in statistical analyses. Canonical correspondence analysis (CCA) determined that pH, ammonium, aluminum, spring total phosphorus (TP), strontium, total nitrogen (TN), maximum depth (MaxZ), chlorophyll a (Chla) and mean depth were significant variables in explaining the variance in the diatom species data. The environmental optima of common diatom taxa for the limnologically important variables (TP, pH, TN, MaxZ, Chla) were calculated using weighted averaging (WA) regression and calibration techniques, and transfer functions were generated. The diatom inference model for spring TP provided a robust reconstructive relationship (r2 = 0.637; RMSE = 0.007 mg L-1; r2boot = 0.466; RMSEboot = 0.010 mg L-1). Other variables, including pH (r2 = 0.702; RMSE = 0.208; r2boot = 0.485; RMSEboot = 0.234), TN (r2 = 0.574; RMSE = 0.0899 mg L-1; r2boot = 0.380; RMSEboot = 0.127 mg L-1) and MaxZ (r2 = 0.554; RMSE = 1.05 m; r2boot = 0.380; RMSEboot = 1.490 m), were also strong, indicating that they may also be reconstructed from fossil diatom communities. This study shows that it is possible to reliably infer lakewater TP and other limnological variables in alkaline Southern Ontario lakes using the WA technique. This method has the potential to aid rehabilitation programs, as it can provide water quality managers with the means to estimate pre-enrichment phosphorus concentrations and an indication of the onset and development of nutrient enrichment in a lake.