Muscle stiffness in the spinal region is essential for maintaining spinal function, and might be related to multiple spinal musculoskeletal disorders. However, information on the distribution of muscle stiffness along the spine in different postures in large subject samples has been lacking, which merits further investigation. This study introduced a new protocol of measuring bilateral back muscle stiffness along the thoracic and lumbar spine (at T3, T7, T11, L1 & L4 levels) with both ultrasound shear-wave elastography (SWE) and tissue ultrasound palpation system (TUPS) in the lying and standing postures of 64 healthy adults. Good inter-/intra-reliability existed in the SWE and TUPS back muscle stiffness measurements (ICC ≥ 0.731, p < 0.05). Back muscle stiffness at the L4 level was found to be the largest in the thoracic and lumbar regions (p < 0.05). The back muscle stiffness of males was significantly larger than that of females in both lying and standing postures (p < 0.03). SWE stiffness was found to be significantly larger in standing posture than lying among subjects (p < 0.001). It is reliable to apply SWE and TUPS to measure back muscle stiffness. The reported data on healthy young adults in this study may also serve as normative reference data for future studies on patients with scoliosis, low back pain, etc.