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Laboratory investigations have provided important insight into the functional underpinnings of primate locomotor performance; however, it is unclear to what extent gait patterns in the laboratory reflect those of primates moving in natural settings. We filmed quadrupedal loco-motor activity in eight platyrrhine species at the Tiputini Biodiversity Station, Ecuador, and three additional platyrrhine species at La Suerte Biological Field Station, Costa Rica, and also quantified the diameter and orientation of locomotor substrates using remote sensors (N = 1,233 strides). We compared overall arboreal quadrupedal gait kinematic patterns in free-ranging individuals to those of laboratory platyrrhine congenerics. As expected, gait kinematics of free-ranging individuals were more variable than laboratory counterparts. Within the free-ranging dataset, Ateles and Alouatta increased limb phase on inclines (p=0.04; p=0.002, respectively), Lagothrix increased duty factors on inclines (p=0.002), Cebus increased duty factors on declines (p=0.02), and both Saimiri and Saguinus displayed an inverse relationship between limb phase and substrate diameter (p=0.05; p=0.03, respectively). This study confirms the preference for diagonal sequence gaits in free-ranging primates (i.e., 87.9% of all recorded symmetrical strides) and that in both settings primates tend to adjust gait patterns to promote security through longer contact times on non-horizontal substrates and increased limb phase on inclined substrates. We show that laboratory and field investigations of primate locomotion yield consistent patterns but that field studies can capture additional aspects of gait variability and flexibility in response to the increased substrate complexity of natural environments.