To measure the distribution of local contractile movements in the layer of the myotubes, the movement of the matrix pattern of micromarkers placed on the back surface of the myotube-attached thin film has been tracked in vitro. A matrix pattern of micromarkers (diameter: 4 μm, height: 2 μm, interval: 30 μm) was fabricated on the back of a polydimethylsiloxane thin film (thickness: 6 μm) using photolithography. Mouse myoblasts were seeded on the front of the thin film and cultured for 10 days to differentiate into myotubes. Periodic electrical pulses (amplitude 2 V, pulse period 0.5 s, pulse width 1 ms) were applied between the electrodes of titanium wires immersed in the medium. Movements of both myotubes and markers were observed simultaneously with an optical microscope. Experimental results indicate that the motion distribution of each marker is related to the local periodic motion of the myotube layer. Thin films with matrix-patterned micromarkers fabricated on the back side allow in vitro analysis of the distribution of local repetitive contractile movements in the myotube layer.