Objective: The validity and reliability of 3-D ultrasound (US) in estimation of muscle and tendon volume was assessed in a very limited number of muscles that can be easily immersed. The objective of the present study was to assess the validity and reliability of muscle volume measurements for all hamstring muscle heads and gracilis (GR), as well as tendon volume for the semitendinosus (ST) and GR using freehand 3-D US. Methods: Three-dimensional US acquisitions were performed for 13 participants in two distinct sessions on separate days, in addition to one session dedicated to magnetic resonance imaging (MRI). Volumes of ST, semimembranosus (SM), biceps femoris short (BFsh) and long (BFlh) heads, and GR muscles and from the tendon from semitendinosus (STtd) and gracilis (GRtd) were collected. Results: The bias and the 95% confidence intervals of 3-D US compared with MRI ranged from -1.9 mL (-0.8%) to 1.2 mL (1.0%) for muscle volume and from 0.01 mL (0.2%) to -0.03 mL (-2.6%) for tendon volume. For muscle volume assessed using 3-D US, intraclass correlation coefficients (ICCs) ranged from 0.98 (GR) to 1.00, and coefficients of variation (CV) from 1.1% (SM) to 3.4% (BFsh). For tendon volume, ICCs were 0.99, and CVs between 3.2% (STtd) and 3.4% (GRtd). Conclusion: Three-dimensional US can provide a valid and reliable inter-day measurement of hamstrings and GR for both muscle and tendon volumes. In the future, this technique could be used as an outcome for strengthening interventions and potentially in clinical environments.