Fish species richness in Ontario lakes was summarized for 80 equal-area quadrats and ranged from 59 species per quadrat in southwestern Ontario to 17 species per quadrat in northern Ontario. Correlograms based on Moran's I coefficient indicated the spatial structure of lake species richness was anisotropic and exhibited a strong south–north gradient of decreasing richness. Relationships among Euclidean distance matrices based on richness, postglacial dispersal, climate, elevation, lake chemistry, and lake morphometry were identified with the aid of the Mantel test and principal coordinates analysis. The presence of spatial autocorrelation may bias matrix correlations; therefore, spatial autocorrelation was removed using the partial Mantel test. The results of the partial Mantel test indicated regional lake species richness was significantly correlated with postglacial dispersal and climate. In particular, the results of principal coordinates analysis identified length of time since deglaciation, length of time covered by glacial lakes, distance from dispersal corridor, and mean annual air temperature as the dominant variables in these data sets.