Understanding the effects of forest fragmentation on tree-dwelling sciurids is of particular interest given their arboreal habits and the extent of anthropogenic habitat fragmentation inflicted upon North American forest ecosystems over the past 2 centuries. In this study we investigate occurrences of northern flying squirrels (Glaucomys sabrinus) and red squirrels (Tamiasciurus hudsonicus) in forest fragments in southern Ontario, Canada, as a function of local habitat and landscape features. During the summer of 2006 we measured occurrence via livetrapping in 24 forest fragments ranging in size from 4 to 2,881 ha, each adjacent to or surrounded by active row-crop agriculture. In addition to patch area and measurements of local habitat features, we calculated 4 landscape metrics in variously sized circular landscape windows: number of patches, forest cover, mean proximity index, and distance to the nearest neighboring patches. Occurrence of G. sabrinus was positively correlated with patch area (P  =  0.016) but not with other features, whereas occurrence of T. hudsonicus was positively associated with basal area of coniferous trees (P  =  0.047) but not with other habitat or landscape features. Populations of T. hudsonicus did not show fragmentation effects, likely due to high vagility and high population growth potential. Northern flying squirrels were not found in patches < 29 ha in size and, as estimated from a receiver operating characteristic curve, the ideal minimum fragment size for patch occupancy was 48.25 ha. Our data support conclusions that diverse management schemes may be required to preserve relatively large contiguous tracts of forest for G. sabrinus and appropriate conifer structure for T. hudsonicus in a way that will facilitate the persistence of these 2 species in deciduous Great Lakes–St. Lawrence forest ecosystems.