Evolution of the human hand has undergone a transition from use during locomotion to use primarily for manipulation. Previous comparative morphological and biomechanical studies have focused on potential changes in manipulative abilities during human hand evolution, but few have focused on functional signals for arboreal locomotion. Here, we provide this comparative context though the first analysis of hand loading in captive bonobos during arboreal locomotion. We quantify pressure experienced by the fingers, palm and thumb in bonobos during vertical locomotion, suspension and arboreal knuckle-walking. The results show that pressure experienced by the fingers is significantly higher during knuckle-walking compared with similar pressures experienced by the fingers and palm during suspensory and vertical locomotion. Peak pressure is most often experienced at or around the third digit in all locomotor modes. Pressure quantified for the thumb is either very low or absent, despite the thumb making contact with the substrate during all suspensory and vertical locomotor trials. Unlike chimpanzees, bonobos do not show a rolling pattern of digit contact with the substrate during arboreal knuckle-walking-instead, we found that digits 3 and 4 typically touch down first and digit 5 almost always made contact with the substrate. These results have implications for interpreting extant and fossilized hand morphology; we expect bonobo (and chimpanzee) bony morphology to primarily reflect the biomechanical loading of knuckle-walking, while functional signals for arboreal locomotion in fossil hominins are most likely to appear in the fingers, particularly digit 3, and least likely to appear in the morphology of the thumb.