Prehensile appendages represent an adaptation that has evolved in various vertebrate and invertebrate lineages. Prehensility is the ability to hold and grasp firmly onto an object, combining flexibility and strength. Chameleons (Chamaeleonidae) typically have an arboreal lifestyle and use their prehensile tails for anchoring and as a support during feeding. By linking the variation in morphology and musculature to function, we aim to explain what it takes to make a tail prehensile. A previous study focused on the morphological variation in tail vertebrae of prehensile and non-prehensile chameleon species using µCT data, however without information on tail musculature this data alone does not allow functional testing and analysis. Our morphological data showed considerable shape variation in the transversal spine between prehensile and non-prehensile species. For this study, we focus on the muscle bundles that attach to the transversal spine, in particular the m. ilio-caudalis, which has an important role in the torsion and ventral flexion of the tail. Both length and angle of the transversal spine differ between prehensile and non-prehensile species, as well as regionally within the tail. Prehensile species have a longer transversal spine pointing distally, that decreases towards the distal end. Studying prehensile function at a musculoskeletal level, we used µCT scans of PMA stained specimens and dissections to reveal where on the spine the muscles attach and how many vertebrae they cross before insertion. Using that muscle data as input with a virtual 3D reconstructions of the tail vertebrae, we used multibody dynamics analysis to investigate the role of the individual muscles on the movement and function of the tail.