Ultrasound (US) is a widely used modality for medical imaging, since it is non-invasive and relatively inexpensive. The ability of US imaging to detect internal structures, like tissue interfaces or lesion sites makes it a promising candidate for dental imaging. So far, the inherent technical difficulties of US imaging in tissues and organs with very heterogeneous (acoustic) properties and limited access have prevented its widespread use. In this study, we characterized the acoustic properties of sectioned teeth by scanning acoustic microscopy in reflection and transmission modes. The spatial distribution of sound velocity was measured in sections of extracted human teeth by use of a scanning acoustic microscope (SAM). Freshly extracted teeth were fixed in 4% formaldehyde solution and embedded in a polymer block (PMMA). Sections of approximately 1 mm thickness were cut along the coronal-apical axis. Radio frequency (RF) data of teeth were collected in a scan region of 15 × 15 mm2 by a SAM operating at 30 MHz with a lateral step size of 50 μm and a sampling rate of 500 MSa/s. Sound velocity was determined from the time resolved reflection and transmission signals. Values for sound velocity from transmission mode were about 20% lower than that from the reflection mode, if thickness information from reflection mode was used. If thickness was determined from the transmission mode, sound velocities from transmission were very close to those obtained from the reflection mode. Transmission mode is less sensitive to artifacts caused by the inclination of the specimen.