Purpose: To advance our current knowledge of singer physiology by using ultrasonography in combination with acoustic measures to compare physiological differences between musical theater (MT) and opera (OP) singers under controlled phonation conditions. Primary objectives addressed in this study were (a) to determine if differences in hyolaryngeal and vocal fold contact dynamics occur between two professional voice populations (MT and OP) during singing tasks and (b) to determine if differences occur between MT and OP singers in oral configuration and associated acoustic resonance during singing tasks. Method: Twenty-one singers (10 MT and 11 OP) were included. All participants were currently enrolled in a music program. Experimental procedures consisted of sustained phonation on the vowels /i/ and /a/ during both a low-pitch task and a high-pitch task. Measures of hyolaryngeal elevation, tongue height, and tongue advancement were assessed using ultrasonography. Vocal fold contact dynamics were measured using electroglottography. Simultaneous acoustic recordings were obtained during all ultrasonography procedures for analysis of the first two formant frequencies. Results: Significant oral configuration differences, reflected by measures of tongue height and tongue advancement, were seen between groups. Measures of acoustic resonance also showed significant differences between groups during specific tasks. Both singer groups significantly raised their hyoid position when singing high-pitched vowels, but hyoid elevation was not statistically different between groups. Likewise, vocal fold contact dynamics did not significantly differentiate the two singer groups. Conclusions: These findings suggest that, under controlled phonation conditions, MT singers alter their oral configuration and achieve differing resultant formants as compared with OP singers. Because singers are at a high risk of developing a voice disorder, understanding how these two groups of singers adjust their vocal tract configuration during their specific singing genre may help to identify risky vocal behavior and provide a basis for prevention of voice disorders.