The two-wave phenomenon, the wave separation of a single ultrasonic pulse in cancellous bone, is expected to be a useful tool for the diagnosis of osteoporosis. However, because actual bone has a complicated structure, precise studies on the effect of transition conditions between cortical and cancellous parts are required. This study investigated how the transition condition influenced the two-wave generation using three-dimensional X-ray CT images of an equine radius and a three-dimensional simulation technique. As a result, any changes in the boundary between cortical part and trabecular part, which gives the actual complex structure of bone, did not eliminate the generation of either the primary wave or the secondary wave at least in the condition of clear trabecular alignment. The results led us to the possibility of using the two-wave phenomenon in a diagnostic system for osteoporosis in cases of a complex boundary.