Purpose To propose a manual segmentation method for individual quadriceps femoris (QF) muscles and to test its reliability for muscle volume estimation. Materials and Methods Images were acquired every 5 mm along the thigh using a 3T MRI scanner on 10 young (mean age: 25 years) and 10 older (mean age: 75 years) adults using a three‐point 3D Dixon sequence. In each slice, anatomical cross‐sectional areas of the individual quadriceps muscles of the dominant leg were outlined by two operators working independently. Differences between operators were assessed by means of Bland–Altman plots and intraclass correlation coefficients (ICC). This study was approved by the local Ethics Committee. Results Precise delimitation of individual muscles along the femur often remains challenging, particularly near their insertion areas where some muscles may be partially or totally fused. There was, however, an excellent interoperator segmentation reliability despite a systematic significant difference between operators (ICC > 0.99), mainly due to delineation divergences. Considering all subjects and muscles, differences between operators were all lower than 4.4%. Conclusion This work has demonstrated the excellent reliability of manual segmentation to assess cross‐sectional areas and therefore the volume of individual QF muscles using MRI. It may serve as a basis for a future segmentation consensus of the QF muscles. J. Magn. Reson. Imaging 2014;40:239–247. © 2013 Wiley Periodicals, Inc.