Abstract Background Time-efficient and accurate whole volume thigh muscle segmentation is a major challenge in moving from qualitative assessment of thigh muscle MRI to more quantitative methods. This study developed an automated whole thigh muscle segmentation method using deep learning for reproducible fat fraction quantification on fat–water decomposition MRI. Results This study was performed using a public reference database (Dataset 1, 25 scans) and a local clinical dataset (Dataset 2, 21 scans). A U-net was trained using 23 scans (16 from Dataset 1, seven from Dataset 2) to automatically segment four functional muscle groups: quadriceps femoris, sartorius, gracilis and hamstring. The segmentation accuracy was evaluated on an independent testing set (3 × 3 repeated scans in Dataset 1 and four scans in Dataset 2). The average Dice coefficients between manual and automated segmentation were > 0.85. The average percent difference (absolute) in volume was 7.57%, and the average difference (absolute) in mean fat fraction (meanFF) was 0.17%. The reproducibility in meanFF was calculated using intraclass correlation coefficients (ICCs) for the repeated scans, and automated segmentation produced overall higher ICCs than manual segmentation (0.921 vs. 0.902). A preliminary quantitative analysis was performed using two-sample t test to detect possible differences in meanFF between 14 normal and 14 abnormal (with fat infiltration) thighs in Dataset 2 using automated segmentation, and significantly higher meanFF was detected in abnormal thighs. Conclusions This automated thigh muscle segmentation exhibits excellent accuracy and higher reproducibility in fat fraction estimation compared to manual segmentation, which can be further used for quantifying fat infiltration in thigh muscles.