Image-guided surgery (IGS) can improve the patient’s outcome by providing meaningful real-time information about the location of cancerous tumors and surrounding tissue, aiding in the elimination of positive tumor margins and reducing iatrogenic damage. However, the clinical need for imaging systems that can provide real-time feedback under real operating room settings remains unmet. State-of-the-art imaging systems for near-infrared fluorescence IGS rely on a series of complex optics and several imaging sensors. As a result, these systems are bulky and expensive, and their architecture lacks the versatility to simultaneously image multiple fluorophores, effectively making them cumbersome when merged into the current surgical workflow. To address these shortcomings, we have designed a multi-spectral imager capable of spatially co-registered hexachromatic vision: three spectral channels in the visible spectrum for the identification of anatomical features in color and three spectral channels in the near-infrared spectrum for the simultaneous identification of multiple near-infrared fluorescence dyes used in IGS. Our single-chip imaging sensor combines the vertically stacked photodetectors technology with pixelated interference filters to create a multi-spectral imager that can help surgeons make clinically relevant decisions in real time, with an effective resolution of 1280x720x3 photodiodes and a frame rate of 24 FPS. Our imager has the ability to identify different shades of near-infrared fluorescent light, allowing the surgeon to use and differentiate multiple fluorophores as molecular probes with high sensitivity. Pre-clinical data is shown where simultaneous imaging of anatomical features in color, and identification of nerves and cancerous tumors, are achieved using multiple near-infrared fluorescent agents.