We present a study to evaluate and compare three near-infrared (NIR) fluorescence imaging systems designed to provide intraoperative guidance around anatomical structures. The three systems adapted specifically for the application of endogenous NIR fluorescence detection were (1) a photomultiplier tube based NIR viewer, (2) a thermoelectrically cooled electron-multiplying charge-coupled device (CCD) camera, and (3) a clinical endoscope CCD camera system. Each system was evaluated on the basis of ease-of-use, cost, and system performance. The cooled CCD camera showed the highest contrast ratio, but is limited in utility by its bulk interface and high cost. The clinical endoscope camera showed flexibility in its field-of-view and provides the benefit of being sterilized to allow easy integration into the surgical suite; however, it exhibits signal nonlinearity that would distort quantitative analysis. The NIR viewer shows optimal performance, exhibiting high spatial resolution, linearity, and sufficient contrast to differentiate between tissue types. This low-cost design proves to be the optimal system for parathyroid detection, offering ease-of-use in a surgical setting while meeting system performance requirements.