Microfluidic‐based technologies enable the development of cell culture systems that provide tailored microenvironmental inputs to mammalian cells. Primary myoblasts can be induced to differentiate into multinucleated skeletal muscle cells, myotubes, which are a relevant model system for investigating skeletal muscle metabolism and physiology in vitro. However, it remains challenging to differentiate primary myoblasts into mature myotubes in microfluidics devices. Here we investigated the effects of integrating continuous (solid) and intermittent (dashed) walls in microfluidic channels as topological constraints in devices designed to promote the alignment and maturation of primary myoblast‐derived myotubes. The topological constraints caused alignment of the differentiated myotubes, mimicking the native anisotropic organization of skeletal muscle cells. Interestingly, dashed walls facilitated the maturation of skeletal muscle cells, as measured by quantifying myotube cell area and the number of nuclei per myotube. Together, our results suggest that integrating dashed walls as topographic constraints in microfluidic devices supports the alignment and maturation of primary myoblast‐derived myotubes. A microfluidic device designed to promote the alignment and maturation of myotubes differentiated from primary myoblasts is described. Song and coworkers investigated the effects of integrating continuous and intermittent walls as topological constraints in microfluidics devices.