Penguin tarsometatarsi are shortened and flattened, and studies devoted to the internal characteristics of these composite bones are very limited. Therefore, we present here a comprehensive, x-ray-microscopy-based analysis based on tarsometatarsi of Eocene stem Sphenisciformes from Seymour Island (Antarctic Peninsula) as well as recent Aptenodytes forsteri, A. patagonicus, and Pygoscelis adeliae penguins. Our study focuses on four aspects: size variability of the medullary cavities, vascularization patterns with emphasis on diaphyseal vessels, cross-sectional anisotropy, and diaphyseal resistance to bending forces. Small-sized Eocene penguins (Delphinornis and Marambiornopsis) show well-developed tarsometatarsal medullary cavities, whereas the cavities of “giant” early Sphenisciformes are either smaller (Palaeeudyptes) or show a conspicuous intermetatarsal size gradient (Anthropornis). Extant penguins exhibit a decrease in cavity dimensions as their body size increases. Distributional tendencies of primary diaphyseal nutrient foramina are quite similar in the smaller Delphinornis, Marambiornopsis, and extant Pygoscelis on one side and in Palaeeudyptes and extant Aptenodytes on the other. Anthropornis shows a unique, plesiomorphic pattern with a prevalence of plantar blood supply to the metatarsals. The diaphyseal nutrient canals diverge in orientation, some obliquely away from the proximal part, others with disparate trajectories. Cross-sectional anisotropy along the tarsometatarsal shaft generally appears to be rather low. Clustering of coherency curves along certain tarsometatarsal segments may reflect a selection process that exerts a significant influence within biomechanically crucial sections. Diaphyseal resistance to mediolateral bending forces is explicitly more efficient in extant penguins than in Eocene Sphenisciformes. This can be interpreted as an adaptation to the waddling gait of extant penguins.