Abstract This study reports the implementation of an endoscope-based near-infrared (NIR) autofluorescence (AF) spectroscopy technique for in vivo differentiation of normal, hyperplastic and adenomatous colonic polyps during clinical colonoscopic examination. A total of 198 in vivo NIR AF spectra in the range of 810–1050 nm were acquired from colonic tissues (normal ( n = 116); hyperplastic ( n = 48); and adenomatous polyps ( n = 34)) of 96 patients undergoing colonoscopic screening. Significant differences ( p < 0.001, one-way analysis of variance (ANOVA)) in in vivo NIR AF intensity among normal, hyperplastic, and adenomatous polyps are observed. Multivariate statistical techniques, including principal components analysis (PCA) and linear discriminate analysis (LDA) together with the leave-one tissue site-out, cross-validation, were used to develop diagnostic algorithms for distinguishing adenomatous polyps from normal and hyperplastic colonic polyps based on NIR AF spectral features. The PCA–LDA modeling on in vivo colonic NIR AF dataset yields diagnostic sensitivities of 83.6%, 77.1%, and 88.2%; and specificities of 96.3%, 88.0%, and 92.1%, respectively, for classification of normal, hyperplastic and adenomatous colonic polyps. This work suggests that NIR AF spectroscopy associated with PCA–LDA algorithms has potential for in vivo diagnosis and detection of colonic precancer at colonoscopy.