In vivo and in vitro studies have shown that high contrast images of tooth demineralization can be acquired in the near-IR due to the high transparency of dental enamel. The purpose of this study is to compare the lesion contrast in reflectance at near-IR wavelengths coincident with high water absorption with those in the visible, the near-IR at 1300-nm and with fluorescence measurements for early lesions in occlusal surfaces. Twenty-four human molars were used in this in vitro study. Teeth were painted with an acid-resistant varnish, leaving a 4×4 mm window in the occlusal surface of each tooth exposed for demineralization. Artificial lesions were produced in the exposed windows after 1 & 2-day exposure to a demineralizing solution at pH 4.5. Lesions were imaged using NIR reflectance at 3 wavelengths, 1310, 1460 and 1600-nm using a high definition InGaAs camera. Visible light reflectance, and fluorescence with 405-nm excitation and detection at wavelengths greater than 500-nm were also used to acquire images for comparison. Crossed polarizers were used for reflectance measurements to reduce interference from specular reflectance. The contrast of both the 24 hr and 48 hr lesions were significantly higher (P<0.05) for NIR reflectance imaging at 1460-nm and 1600-nm than it was for NIR reflectance imaging at 1300-nm, visible reflectance imaging, and fluorescence. The results of this study suggest that NIR reflectance measurements at longer near-IR wavelengths coincident with higher water absorption are better suited for imaging early caries lesions.