This dataset includes information about valley segment and catchment summaries, valley characteristics, instream habitat, and fish for valley segments, sites, and transects along four river mainstems in Ontario, Canada. Moving west to east, the rivers include the Grand River which ends in Lake Erie at Port Maitland, the Ganaraska River which ends in Lake Ontario at Port Hope, the Trent River which ends in the Bay of Quinte at Trenton, and the Petawawa River which ends in the Ottawa River at Petawawa. These rivers vary in natural character, anthropogenic development, and fish assemblages. Riverine sites along the mainstems of all four rivers included a total of one hundred and twelve sites. Sampling on the Grand, Trent, and Petawawa Rivers focused on non-wadeable lower river mainstems, whereas all sites on the Ganaraska River mainstem were wadeable and incorporated a wider range of stream sizes. Sites were sampled between 1997 and 2001, with many sites sampled in multiple years. The study design for the Grand, Trent, and Petawawa Rivers include a hierarchical design where data collection was nested at three spatial scales -- shoreline and channel transect data are nested within sites, and sites are nested within valley segments. In the Ganaraska River, data collection was by site and nested within valley segments. A complementary set of shapefiles for each river supports these tabular data and provides items needed to map watersheds, valley segments, and sites, and to calculate additional variables for sites and site catchments. The metadata specific to these spatial data is associated with the shapefiles and is not described here.