Abstract Assessment of regional muscle architecture is primarily done through the study of animals, human cadavers, or using b‐mode ultrasound imaging. However, there remain several limitations to how well such measurements represent in vivo human whole muscle architecture. In this study, we developed an approach using diffusion tensor imaging and magnetic resonance imaging to quantify muscle fibre lengths in different muscle regions along a muscle's length and width. We first tested the between‐day reliability of regional measurements of fibre lengths in the medial (MG) and lateral gastrocnemius (LG) and found good reliability for these measurements (intraclass correlation coefficient [ICC] = 0.79 and ICC = 0.84, respectively). We then applied this approach to a group of 32 participants including males ( n = 18), females ( n = 14), young (24 ± 4 years) and older (70 ± 2 years) adults. We assessed the differences in regional muscle fibre lengths between different muscle regions and between individuals. Additionally, we compared regional muscle fibre lengths between sexes, age groups, and muscles. We found substantial variability in fibre lengths between different regions within the same muscle and between the MG and the LG across individuals. At the group level, we found no difference in mean muscle fibre length between males and females, nor between young and older adults, or between the MG and the LG. The high variability in muscle fibre lengths between different regions within the same muscle, possibly expands the functional versatility of the muscle for different task requirements. The high variability between individuals supports the use of subject‐specific measurements of muscle fibre lengths when evaluating muscle function.