Intramuscular color stability variations in beef semimembranosus have been reported previously. Mitochondria remain biochemically active in postmortem muscle and can influence fresh beef color stability. However, the role of mitochondrial functionality in intramuscular color variations in beef semimembranosus is yet to be examined. We examined the functionality of mitochondria isolated from outside (OSM) and inside (ISM) regions of beef semimembranosus. Semimembranosus muscles (n = 5) were collected from inside rounds of beef carcasses 48 h post-mortem and were separated to OSM and ISM steaks. Color attributes were evaluated instrumentally and biochemically on days 0 and 4 of retail display, whereas mitochondrial oxygen consumption rate (OCR) was measured on day 0 using succinate as substrate using steaks frozen during fabrication. Mitochondrial OCR was significantly greater in OSM than in ISM on day 0. The ISM steaks exhibited significantly greater redness (a* value) than OSM steaks on day 0, but OSM steaks had significantly greater redness than the ISM counterparts on day 4. During retail display, ISM steaks exhibited greater lightness (L* value) than OSM steaks. However, OSM demonstrated significantly greater color stability and metmyoglobin reducing activity than ISM throughout the display. The observed differences in mitochondrial OCR between ISM and OSM steaks indicated that mitochondrial biochemistry possibly contributes to the intramuscular color variations in beef semimembranosus.