This paper presents a novel segmentation methodology for automated classification and differentiation of soft tissues using multiband data obtained with the newly developed system of high-resolution ultrasonic transmission tomography (HUTT) for imaging biological organs. This methodology extends and combines two existing approaches: the L-level set active contour (AC) segmentation approach and the agglomerative hierarchical kappa-means approach for unsupervised clustering (UC). To prevent the trapping of the current iterative minimization AC algorithm in a local minimum, we introduce a multiresolution approach that applies the level set functions at successively increasing resolutions of the image data. The resulting AC clusters are subsequently rearranged by the UC algorithm that seeks the optimal set of clusters yielding the minimum within-cluster distances in the feature space. The presented results from Monte Carlo simulations and experimental animal-tissue data demonstrate that the proposed methodology outperforms other existing methods without depending on heuristic parameters and provides a reliable means for soft tissue differentiation in HUTT images.