The tongue is a muscular hydrostat whose complex fibre architecture enables its diverse functions in swallowing, speech, and breathing. Current understanding of the tongue's structure is largely based on ex vivo dissections, which are not directly linked to function. This study aimed to develop an anatomical atlas of the living human tongue incorporating detailed muscle architecture. The pharynx of 20 healthy volunteers (10 females) were imaged with 3T MRI, collecting mDIXON and diffusion-weighted images (DWI). Multichannel registration was used to align scans from individual participants into a common spatial reference frame to create a population-averaged tongue atlas. The atlas was able to reliably predict tongue muscle architecture of tongues not used in atlas construction, although accuracy varied with the image types used. The best performance, assessed with a local angular correlation coefficient (LACC), was achieved when both anatomical (mDIXON) and diffusion-weighted images were used (LACC = 0.66 ± 0.04
p ≪ 0.001), but acceptable accuracy was achieved when only anatomical images were used (LACC = 0.52 ± 0.04
p ≪ 0.001). Principal component analysis of a tongue statistical shape model based on the atlas identified that the largest source of variation in tongue muscle architecture was related to the position of the hyoid relative to the mandibular plane. A lower hyoid was more common in males (M: 13 mm, F: 9 mm
p = 0.009). This new atlas of in vivo tongue muscle architecture provides a new understanding of the relationships between the muscles and bony structures that may enable more accurate simulation of human living tongue function.