We examined long-term changes in phytoplankton composition from 1981 to 2003 in seven intensively studied lakes on the southern Canadian Shield in Ontario, Canada. Significant (P < 0.05), temporally coherent increases in the relative biovolume of colonial chrysophytes were observed in six of the seven lakes, with coincident declines in the relative biovolume of diatom algae. Variance partitioning analyses identified water chemistry variables, and the co-variation of water chemistry with physicoclimatic variables, as most important in structuring phytoplankton assemblages through time in the study lakes (variance explained: chemical variables (14%–47%, mean = 28.7%); chemistry and physicoclimatic variables (21%–30%, mean = 25.5%)). With the exception of Harp Lake, which was invaded by Bythotrephes in the early 1990s, grazing variables did not explain a significant portion of the phytoplankton variance. We hypothesize that the long-term changes in phytoplankton species composition is attributable to multiple anthropogenic stressors acting at a regional scale. Our results, coupled with paleoecological studies, indicate that increases in the relative importance of colonial chrysophytes are coincident with water chemistry changes associated with industrial activity since the mid-1900s and physical changes linked to climate indices such as the North Atlantic Oscillation.