Detailed analysis of skeletal muscle architecture provides insights into skeletal muscle function. To date, measurements of the human subscapularis architecture have been limited to cadaveric measurements. In this study we demonstrate the feasibility of using anatomically constrained fibre tractography to reconstruct and quantify the 3D architecture of the human subscapularis muscle, and provide the first quantitative measurements of the architecture of the human subscapularis muscle in vivo. mDixon and diffusion tensor magnetic resonance images were obtained from the right shoulders of 20 healthy young adults. Anatomically constrained fibre tractography, in which fascicle reconstructions were forced to terminate on the internal aponeurosis of the subscapularis, was used to reconstruct muscle fibre architecture of the subscapularis muscles. Qualitatively, architectural reconstructions resembled the known subscapularis anatomy well, demonstrating face validity of the reconstructions. Muscle architectural parameters (means ± SDs) were:​ muscle volume 138 ± 42 cm³, fascicle length 63.6 ± 5.9 mm, physiological cross-sectional area (PCSA) 22 ± 6 cm², and pennation angle 16 ± 2°. Architectural measurements of the subscapularis fell within the range reported in cadaver studies and were relatively insensitive to variations in fibre tractography parameters. The anatomically detailed whole-muscle reconstructions can be used to quantify the effects of joint surgery on muscle architecture and to advance computational models of the human shoulder.