High-frequency ultrasound techniques are introduced for three-dimensional imaging and thickness estimation of fresh heart valve cusps. Images of porcine aortic valve specimens were acquired within a 12 × 8 × 8 mm³ volume using a VisualSonics VS40 micro-imaging system operating at a 40 MHz centre frequency. Two image volumes were obtained from each of six left coronary cusps. One volume was acquired with the specimen submerged in distilled water and the second volume was acquired through either Hanks physiologic solution or coronary perfusion solution (CPS). The fibrosa, spongiosa and ventricularis were most readily distinguished when the specimen was imaged in distilled water. Colour thickness maps were computed from B-mode image data, and the mean and standard deviations of the thickness were determined for each cusp. In 11 of 12 trials, the image analysis algorithm yielded valid thickness estimates over greater than 98% of the region examined. Mean thickness estimates obtained with specimens submerged in Hanks solution or CPS ranged from 0.66 to 1.03 mm, and submersion in distilled water increased the mean thickness by 20–40%. This observation suggests that the cusps osmotically absorbed water. Information provided by high-frequency ultrasound is expected be valuable for characterizing the morphological properties of heart valves.