Diagonal-sequence (DS) gaits, which are very rare among mammals, are common and well documented in primates and some arboreal marsupials. DS walking gaits have been reported in the kinkajou (Potos flavus), which shows ecological similarities with primates and arboreal opossums but lacks prehensile specializations of the hindfoot. Nevertheless, the actual frequency of DS gaits and the functional context in which these gaits occur in this highly arboreal mammal remain unknown. We examined the effect of substrate size on the locomotion and gait patterns of kinkajous by recording gaits in two individuals walking and running on poles of two different diameters and on a runway. Diagonality and limb duty factors were calculated for 534 gait cycles. Kinkajous relied mostly on DS gaits and trots during walking, and increased the diagonality of their gait patterns on thinner substrates. The proposed functional link between locomotion on thin branches and the presence of a grasping, primate-like hindfoot is not supported by these data. However, further analysis of kinkajou gait cycles showed that DS gaits may have advantages overlooked earlier. DS gaits, during walking, minimize the distance between two ipsilateral feet during short periods of unilateral bipedality, and per corollary maximize the distance between two contralateral feet during the much longer periods of diagonal bipedality. Such foot positioning during the gait cycle could be beneficial in walking on a relatively thin substrate and could explain why kinkajous adopt DS walking gaits, especially on thinner poles, despite lacking prehensile specializations of the hindfoot. J. Exp. Zool. 313A:157–168, 2010. © 2010 Wiley-Liss, Inc.