The relationship between collagen fibril morphology and the functional behavior of tendon tissue has been investigated in numerous experimental studies. Several of these studies suggest that larger fibril radius is a primary determinant of higher tendon stiffness and strength; others have shown that factors apart from fibril radius (such as fibril–fibril interactions) may be critical to improved tendon strength. In the present study, we investigate these factors in two inbred mouse strains that are widely used in skeletal structure–function research: C57BL/6J (B6) and C3H/HeJ (C3H). The aim was to establish a quantitative baseline that will allow one to assess how regulation of tendon extracellular matrix architecture affects tensile mechanical properties. We specifically focused on collagen fibril structure and glycosaminoglycan (GAG) content – the two primary constituents of tendon by dry weight – and their potential functional interactions. For this purpose, Achilles tendons from both groups were tested to failure in tension. Tendon collagen morphology was analyzed from transmission electron microscopy images of tendon sections perpendicular to the longitudinal axis. Our results showed that the two inbred strains are macroscopically similar, but C3H mice have a higher elastic modulus (P < 0.05). Structurally, C3H mice showed a larger collagen fibril radius compared to B6 mice (96 ± 7 nm and 80 ± 10 nm respectively). Tendons from C3H mice also showed smaller specific fibril surface (0.015 ± 0.001 nm nm−2 vs. 0.017 ± 0.003 nm nm−2 in the B6 tendons, P < 0.05), and accordingly a lower concentration of GAGs (0.60 ± 0.07 μg mg−1 vs. 0.83 ± 0.11 μg mg−1, P < 0.05). As in other studies of tendon structure and function, larger collagen fibril radius appears to be associated with stiffer tendon, but this functional difference could also be attributed to reduced potential surface area exchange between fibrils and the surrounding proteoglycan-rich matrix, in which the hydrophilic GAG side chains may promote inter-fibril sliding. This study provides an architectural and functional baseline for a comparative murine model that can be used to investigate the genetic regulation of tendon biomechanics.