Complex motion of the human thumb is enabled by the balanced architectural design of the extrinsic and intrinsic thumb muscles. Given that recent imaging advances have not yet been applied to enhance our understanding of the in vivo properties of thumb muscles, the objective of this study was to test the reliability and validity of measuring thumb muscle fascicle lengths using extended field of view ultrasound (EFOV-US). Three muscles (FPL:​ flexor pollicis longus, APB:​ abductor pollicis brevis, and ECU:​ extensor carpi ulnaris) were imaged in eight healthy adults (4 female;​ age, 21.6 ± 1.3 years;​ height, 175.9 ± 8.3 cm)[mean ± SD]. Measured fascicle lengths were compared to cadaveric data (all muscles) and ultrasound data (ECU only). Additionally, to evaluate how fascicle lengths scale with anthropometric measurements, height, forearm length, hand length, and hand width were recorded. The EFOV-US method obtained precise fascicle length measurements [mean ± SD] for the FPL (6.2 ± 0.5 cm), APB (5.1 ± 0.3 cm), and ECU (4.0 ± 0.4 cm). However, our EFOV-US measurements were consistently different (p < 0.05) than prior cadaveric data, highlighting the need to better understand differences between in vivo and ex vivo fascicle length measurements. Fascicle length was significantly related to only hand length (r² = 0.56, p = 0.03) for APB, highlighting that anthropometric scaling may not accurately estimate thumb muscle length. As the first study to apply EFOV-US to measure thumb muscle fascicle lengths, this study expands the utility of this imaging technology within the upper limb.