The thyroarytenoid muscle, a vocal fold adductor, has important roles in airway protection (i.e, prevention of aspiration) and phonation. Isoform expression of myosin heavy chain (MHC), a major determinant of muscle shortening velocity, has been reported to be heterogeneous in this muscle among several mammals, differing markedly between the medial and lateral divisions. The objective was to determine the isoform expression patterns of both MHC and myosin light chain (MLC), the latter having a modulatory role in determining shortening velocity, to further test whether the expressions of both myosin subunits differ in multiple specific sites within the divisions of the dog thyroarytenoid muscle, potentially revealing even greater compartmentalization in this muscle. Results indicate that there are large gradients in the relative levels of individual MHC isoforms in the cranial-caudal axis along the medial layer (i.e., airflow axis), where levels of MHC-I and MHC-IIA are low at both ends of the axis and high in the middle, and MHC-IIB has a reciprocal distribution. The lateral layer is more uniform, with MHC-IIB at high levels throughout. The level of MHC-IID is relatively constant along the axis in both layers. There are large differences in the distribution of MHC isoforms among single fibers, isolated from sites along the cranial-caudal axis, especially in the lateral layer. There are systematic regional variations in MLC isoform composition of single fibers as well, including some MLC isoform combinations that are not observed in dog limb muscles. Variations of MHC and MLC isoform expression in the dog thyroarytenoid muscle are greater than previously recognized and suggest that an even broader range of contractile properties exists within this multifunctional muscle.