The production of biotremors has been described in veiled chameleons (Chamaeleo calyptratus), but the mechanism by which they are produced is unknown. We gathered muscle activation data via electromyography (EMG) with simultaneous recordings of biotremors using an accelerometer to test for the role of hyoid muscles in their production. We recorded mean biotremor frequency of 150.87 Hz for females and 136.01 Hz for males. The durations of activity and the latencies to onset and offset for the M. sternohyoideus profundus (SP), M. sternohyoideus superficialis (SS), Mm. mandibulohyoideus (MH), and Mm. levator scapulae (LS) were all significantly correlated with biotremor durations and biotremor onset and offset, respectively. Linear mixed-effect regression model comparisons of biotremor duration indicate that models containing either MH and/or SP and LS account or the most variation in the biotremor duration. Twitch times for the SP (100 ms) and the SS (132 ms) at field active body temperature, however, were individually too slow to produce the biotremors at the observed frequency without alteration after production by other anatomical structures. These results implicate the SP, SS, MH, and LS in the production of the biotremor, but the exact mechanism of production requires further study.