Since 1883, 64 fish species belonging to 22 families have been reported from the north-flowing section of the Rideau River-Canal system, Ontario, Canada. Six of these species are non-native, two of which have become established with the remaining four no longer present. A total of 34 species belonging to 11 families were collected in the mainstem north-flowing section of the Rideau River and five of its tributaries during our 1998–2000 study. The fish community along the Rideau River mainstem at sites with adjacent urban land use was distinguishable from sites that were adjacent to agricultural–forested areas. The urban sites cluster was characterized by an abundance of Rock Bass (Ambloplites rupestris) and a low abundance of Bluegill (Lepomis macrochirus), Pumpkinseed (Lepomis gibbosus), Yellow Perch (Perca flavescens), Brown Bullhead (Ameiurus nebulosus), and White Sucker (Catostomus commersonii). The two mixed agricultural–forested site clusters combined were characterized by an abundance of Black Crappie (Pomoxis nigromaculatus), Largemouth Bass (Micropterus nigricans), Bluegill, Pumpkinseed, Yellow Perch, and Brown Bullhead. Our study is a baseline of the fish community in the Rideau River watershed at the end of the 20th century, with which to compare as the impacts of climate and land use changes continue.