A multi-proxy paleolimnological survey was performed on 13 lakes in the Hudson Bay Lowlands (HBL) of northern Ontario in order to provide a regional analysis of recent environmental changes in this poorly studied sub-Arctic region. In contrast to the amplified warming experienced by most of the circumpolar Arctic since the mid-19th century, the climate of the Hudson Bay (HB) region has remained relatively cool and stable for hundreds of years. However, since approximately the 1990s, the HBL has experienced rapid and large increases in air temperature and declines in sea ice. Diatom, cladoceran, and chironomid remains preserved in the recent (surface) and pre-1850 sediments of 13 lakes were used to examine whether this new climate regime has resulted in species assemblage changes across multiple trophic levels. Our results indicate clear limnological responses to warming among the freshwater biota of HBL lakes; however, the magnitude of this change varied among both biological indicators and sites. As expected, diatoms exhibited the greatest degree of change, closely followed by chironomids, with relatively little change observed among cladoceran assemblages. Planktonic diatoms were more common in modern assemblages, often including plankters that were previously not recorded in the bottom sediments, and in fact all indicator groups recorded a change in benthic/littoral taxa in the recent sediments indicative of warming-induced increases in habitat availability due to decreased lake ice cover.