Objective To evaluate the in vitro rheometric properties of the canine vocal fold lamina propria and muscle at phonatory frequencies, and their changes with anatomic location. Methods Six canine larynges were harvested immediately postmortem. Viscoelastic shear properties of anterior, middle, and posterior portions of the vocal fold cover (lamina propria) as well as those of the medial thyroarytenoid (TA) muscle (vocalis muscle) were quantified by a linear, controlled-strain simple-shear rheometer. Measurements of elastic shear modulus ( G′) and dynamic viscosity ( η′) of the specimens were conducted with small-amplitude sinusoidal shear deformation over a frequency range of 1–250 Hz. Results All specimens showed similar frequency dependence of the viscoelastic functions, with G′ gradually increasing with frequency and η′ decreasing with frequency monotonically. G′ and η′ of the canine vocalis muscle were significantly higher than those of the canine vocal fold cover, and η′ of the canine vocal fold cover was significantly higher than that of the human vocal fold cover. There were no significant differences in G′ and in η′ between different portions of the canine vocal fold cover. Conclusion These preliminary data based on the canine model suggested that the vocalis muscle, while in a relaxed state in vitro, is significantly stiffer and more viscous than the vocal fold cover during vibration at phonatory frequencies. For large-amplitude vocal fold vibration involving the medial portion of the TA muscle, such distinct differences in viscoelastic properties of different layers of the vocal fold should be taken into account in multi-layered biomechanical models of phonation.