The rodent family Sciuridae (squirrels) presents an ideal system for examining the morphological, behavioural and ecological correlates of locomotor novelty. Ancestrally, sciurids were arboreal, a condition retained by modern tree squirrels. Two major transitions from the arboreal condition are hypothesized to have occurred in the sciurid clade: (1) flying squirrels evolved gliding from arboreal leaping (parachuting) and (2) chipmunks and ground squirrels became semiarboreal/terrestrial. This study examines morphology, locomotor behaviour and microhabitat use under controlled laboratory conditions in three North American sciurids: eastern chipmunk Tamias striatus, red squirrel Tamiasciurus hudsonicus and southern flying squirrel Glaucomys volans. Multivariate space was defined using a series of morphological measurements and by continuously sampling individuals moving under identical conditions in a naturalistic enclosure, controlling for the proximate effects of microhabitat structure. Morphospace was characterized by a contrast between proximal and distal limb elements – flying squirrels exhibited elongated forelimbs and shanks and shortened forefeet and hindfeet, while chipmunks exhibited the opposite pattern. Ethospace was characterized by a contrast between aerial locomotion in flying squirrels and ground-based locomotion in chipmunks. In both instances, red squirrels occupied intermediate positions, as predicted on the basis of retention of ancestral features. Despite significant differences among species at morphological and behavioural levels, ecospace was characterized by overlap between flying squirrels and red squirrels in the use of high supports, and between flying squirrels and chipmunks in the use of large-diameter supports. The lack of concordance at the ecological level underscores the difficulty in making predictions based solely upon organismal design.