Introduction: There is no standardized method for examination of facial muscles with ultrasound. The purpose of this study was to identify those facial muscles accessible for reliable identification and to provide reference data. Methods: In healthy subjects all facial muscles were screened for visibility, separation from adjacent muscles, and reliability of landmarks. Bilateral scans of reliable muscles were performed in 40 adult volunteers. Results: Six facial muscles were clearly demarcated with ultrasound. These were: frontalis, orbicularis oculi, orbicularis oris, depressor anguli oris, depressor labii inferioris, and mentalis muscles. Cross‐sectional area and muscle thickness showed gender differences and were independently related to age for some muscles. A significant left–right side difference was only seen for the orbicularis oculi muscle in women. Conclusions: These data demonstrate the usefulness of ultrasonography to assess facial muscles and provide reference values that can be applied in the clinical setting. Muscle Nerve 47: 878–883, 2013