Understanding the structural and functional development of human-induced pluripotent stem-cell-derived cardiomyocytes is essential to engineering cardiac tissue that enables pharmaceutical testing, modeling diseases, and designing therapies. Here, we used a method not commonly applied to biological materials, small angle X-ray scattering to characterize the structural development of human induced pluripotent stem-cell-derived cardiomyocytes within 3D engineered tissues during their preliminary stages of maturation. An innovative X-ray scattering experimental setup enabled the visualization of a systematic variation in the cardiomyocyte myofilament spacing with maturation time. The myofilament lattice spacing monotonically decreased as the tissue matured from its initial post-seeding state over the span of ten days. Visualization of the spacing at a grid of positions in the tissue provides a new approach to characterizing the maturation and organization of cardiomyocyte myofilaments and has the potential to help elucidate mechanisms of pathophysiology, disease progression, thereby stimulating new biological hypotheses in stem cell engineering.