Objective: To assess (1) if fitness and mobility are related to behavior and perception of physical barriers and (2) if behavior and physical barrier perception are related. Design: Cross-sectional case series. Setting: Academic Medical Laboratory. Participants: Manual wheelchair users (N=50) with chronic spinal cord injury (62% paraplegia). Intervention: None. Main Outcome Measures: Participants completed the following assessments: (1) fitness: graded exercise test (aerobic) and Wingate (anaerobic); (2) mobility: 6-minute push test and 30-second sprint test; (3) physical barrier behavior: Encounters of Environmental Features in the Environmental Aspects of Mobility Questionnaire (EAMQ); (4) physical barrier perception: Craig Hospital Inventory of Environmental Factor (CHIEF) Environmental Barriers domain. Results: Individuals with paraplegia had higher fitness, mobility, and environmental barrier encounter rates and lower avoidance per encounter rates vs tetraplegia (all P≤.05). For individuals with tetraplegia only, as mobility and fitness increased, frequencies of (1) encounters increased; (2) avoidances per encounter decreased, in multiple EAMQ domains (all P≤.05). Perception of barriers did not differ between lesion levels (P=.79). Mobility and fitness were not related to environmental barriers perception in both groups (all P>.17). Conclusions: Fitness and mobility are associated with barrier behaviors (ie, encounters and avoidances) among individuals with tetraplegia, but not paraplegia. Despite a greater barrier avoidance rate, persons with tetraplegia do not perceive more physical barriers than persons with paraplegia. Surprisingly, fitness and mobility were not related to perception of barriers in either group. More research is required on if barrier perception, behavior, or both influence participation, to enable rehabilitation programs to tailor interventions to enhance participation.