Near-infrared (NIR) spectroscopic imaging of wounds has been performed by past researchers to obtain tissue oxygenation at discrete point locations. We had developed a near-infrared optical scanner (NIROS) that performs noncontact NIR spectroscopic (NIRS) imaging to provide 2D tissue oxygenation maps of the entire wounds. Regions of changed oxygenation have to be demarcated and registered with respect to visual white light images of the wound. Herein, a semi-automatic image segmentation and co-registration approach using machine learning has been developed to differentiate regions of changed tissue oxygenation. A registration technique was applied using a transformation matrix approach using specific markers across the white light image and the NIR images (or tissue oxygenation maps). This allowed for physiological changes observed from hemodynamic changes to be observed in the RGB white light image as well. Semi-automated segmentation techniques employing graph cuts algorithms was implemented to demarcate the 2D tissue oxygenation maps depicting regions of increased or decreased oxygenation and further coregistered onto the white light images. The developed registration technique was validated via phantom studies (both flat and curved phantoms) and in-vivo studies on controls, demonstrating an accuracy >97%. The technique was further implemented on wounds (here, diabetic foot ulcers) across weeks of treatment. Regions of decreased oxygenation were demarcated, and its area estimated and co-registered in comparison to the clinically demarcated wound area. Future work involves the development of automated machine learning approaches of image analysis for clinicians to obtain real-time co-registered clinical and subclinical assessments of the wound.