Objectives Trabecular microstructure of limb bone epiphyses has been used to elucidate the relationship between skeletal form and behavior among mammals. Such studies have often relied on the analysis of a single volume of interest (VOI). Here we present a method for evaluating variation in bone microstructure across articular surfaces by leveraging sliding semilandmarks. Methods Two samples were used to demonstrate the proposed methodology and test the hypothesis that microstructural variables are homogeneously distributed: tali from two ape genera (Pan and Pongo, n = 9) and modern human distal femora (n = 10). Sliding semilandmarks were distributed across articular surfaces and used to locate the position of multiple VOIs immediately deep to the cortical shell. Trabecular bone properties were quantified using the BoneJ plugin for ImageJ. Nonparametric MANOVA tests were used to make group comparisons and differences were explored using principal components analysis and visualized using color maps. Results Tests reveal that trabecular parameters are not distributed homogeneously and identify differences between chimpanzee and orangutan tali with regards to trabecular spacing and degree of anisotropy, with chimpanzee tali being more anisotropic and having more uniformly spaced trabeculae. Human males and females differed in the pattern of trabecular spacing with males having more uniform trabecular spacing across the joint surface. Conclusions The proposed procedure quantifies variation in trabecular bone parameters across joint surfaces and allows for meaningful statistical comparisons between groups of interest. Consequently it holds promise to help elucidate links between trabecular bone structure and animal behavior.