A novel imaging system for High-resolution Ultrasonic Transmission Tomography (HUTT) and soft tissue differentiation methodology for the HUTT system are presented. The critical innovation of the HUTT system includes the use of sub-millimeter transducer elements for both transmitter and receiver arrays and multi-band analysis of the first-arrival pulse. The first-arrival pulse is detected and extracted from the received signal (i.e., snippet) at each azimuthal and angular location of a mechanical tomographic scanner in transmission mode. Each extracted snippet is processed to yield a multi-spectral vector of attenuation values at multiple frequency bands. These vectors form a 3-D sinogram representing a multi-spectral augmentation of the conventional 2-D sinogram. A filtered backprojection algorithm is used to reconstruct a stack of multi-spectral images for each 2-D tomographic slice that allow tissue characterization. A novel methodology for soft tissue differentiation using spectral target detection is presented. The representative 2-D and 3-D HUTT images formed at various frequency bands demonstrate the high-resolution capability of the system. It is shown that spherical objects with diameter down to 0.3㎜ can be detected. In addition, the results of soft tissue differentiation and characterization demonstrate the feasibility of quantitative soft tissue analysis for possible detection of lesions or cancerous tissue.