Low back and lower extremity injuries are responsible for the highest percentage of musculoskeletal injuries in U.S. Army soldiers. Execution of common soldier tasks as well as army combat fitness test events such as the three-repetition maximum deadlift depends on healthy functioning trunk and lower extremity musculature to minimize the risk of injury. To assist with appropriate return to duty decisions following an injury, reliable and valid tests and measures must be applied by military health care providers. Myotonometry is a noninvasive method to assess muscle stiffness, which has demonstrated significant associations with physical performance and musculoskeletal injury. The aim of this study is to determine the test-retest reliability of myotonometry in lumbar spine and thigh musculature across postures (standing and squatting) that are relevant to common soldier tasks and the maximum deadlift. Repeat muscle stiffness measures were collected in 30 Baylor University Army Cadets with 1 week between each measurement. Measures were collected in the vastus lateralis (VL), biceps femoris (BF), lumbar multifidus (LM), and longissimus thoracis (LT) muscles with participants in standing and squatting positions. Intraclass correlation coefficients (ICCs3,2) were estimated, and their 95% CIs were calculated based on a mean rating, mixed-effects model. The test-retest reliability (ICC3,2) of the stiffness measures was good to excellent in all muscles across the standing position (ICCs: VL = 0.94 [0.87-0.97], BF = 0.97 [0.93-0.98], LM = 0.96 [0.91-0.98], LT = 0.81 [0.59-0.91]) and was excellent in all muscles across the squatting position (ICCs: VL = 0.95 [0.89-0.98], BF = 0.94 [0.87-0.97], LM = 0.96 [0.92-0.98], LT = 0.93 [0.86-0.97]). Myotonometry can reliably acquire stiffness measures in trunk and lower extremity muscles of healthy individuals in standing and squatting postures. These results may expand the research and clinical applications of myotonometry to identify muscular deficits and track intervention effectiveness. Myotonometry should be used in future studies to investigate muscle stiffness in these body positions in populations with musculoskeletal injuries and in research investigating the performance and rehabilitative intervention effectiveness.