ObjectivesThe aim of the present study was to assess the reliability of aponeurosis and muscle thickness of the hamstrings using ultrasound (US).MethodsUS images were captured from the semitendinosus (ST) and biceps femoris long head (BFlh) of 30 individuals (15 females), undergoing two testing sessions with a 24‐hour interval. Measurements were taken at six sites along the thigh at two knee angles (0° = full extension and 90°) in prone position. Aponeurosis and muscle thickness across the entire length of ST and BFlh were evaluated using computational image segmentation which generated ~360 data points per participant.ResultsThe intraclass correlation coefficients ranged from 0.937 to 0.998 while the standard error of measurement varied from 0.88 to 2.72% for superficial aponeurosis, from 1.41 to 2.29% for deep aponeurosis, and from 1.50 to 4.22% for muscle thickness. The minimal detectable change ranged from 2.44 to 7.56% for the superficial aponeurosis, from 3.57 to 6.27% for deep aponeurosis, and from 4.17 to 11.70% for BF and ST muscle thickness.ConclusionsEvaluation of hamstring aponeurosis and muscle thickness measurements at rest displayed very high reliability and, hence, such measurements can be used to diagnose changes in thickness due to injury or exercise interventions.