The objectives of this study were to evaluate longitudinal and lateral variations in color stability and sensory characteristics of the semimembranosus from each muscle location (dorsal=D, medial=M, ventral=V), and within each steak subsequently divided into four regions (caudal-distal=CaD, cranial-distal=CrD, caudal-proximal=CaP, cranial-proximal=CrP). Instrumental color assessment indicated that L(∗) (lightness) values tended to be lower for the CaD and CrD regions of all muscle locations, with higher L(∗) values recorded for the CrP regions of muscle sections. Instrumental tenderness measurements for Meullenet-Owens razor and Warner-Bratzler shear force values were generally lower for the CaD region of the D section, whereas regions within the V section were tougher and required greater shear force. Sensory panelists perceived lower amounts of connective tissue in the CaD of the D section, which tended to be more tender, whereas higher levels of connective tissue and greater toughness occurred in the CaP and CrP regions of the M and V sections. These results provide a detailed muscle profile of the quality variation within the semimembranosus, and may be used to further enhance the fabrication and marketability of this beef subprimal cut.