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Background & Aims Colorectal cancer surveillance guidelines rely on clinicohistologic features of adenomas. Unfortunately, in common practice, recording of these features lacks precision and uniformity, which might hamper appropriate follow-up decisions. Confocal laser endomicroscopy (CLE) is a novel technology that allows real-time in vivo microscopy of the mucosa and provides accurate histopathology. The aims of this study were (1) to define and validate differential features of adenomatous and nonadenomatous colorectal polyps by chromoendoscopy-guided CLE (C-CLE) and (2) to assess predictive value of this technique for diagnosis of colorectal neoplasia. Methods Patients at risk for colorectal cancer were prospectively investigated by using CLE. During extubation, fluorescein 10% was used in conjunction with acriflavine hydrochloride 0.05% to characterize global tissue architecture as well as cytonuclear features of colorectal epithelium. Ex vivo histology was used as gold standard. Reproducibility tests were performed. Results In total, 116 colorectal polyps from 72 patients were examined. Ex vivo histology showed 68 adenomas, 6 invasive carcinomas, 30 hyperplastic polyps, and 12 inflammatory polyps. C-CLE of adenomas revealed lack of epithelial surface maturation, crypt budding, altered vascular pattern, and loss of cell polarity. In contrast, C-CLE of nonadenomatous polyps revealed epithelial surface maturation, and minor abnormalities of crypt architecture and of vascular pattern, and maintained cell polarity. Adenoma dysplasia score reliably discriminated high-grade dysplasia from low-grade dysplasia (accuracy, 96.7%). Interobserver agreement was high (K coefficients: pathologist, 0.92; endomicroscopist, 0.88). In vivo histology predicted ex vivo data with sensitivity of 97.3%, specificity of 92.8%, and accuracy of 95.7%. Conclusions C-CLE accurately discriminates adenomatous from nonadenomatous colorectal polyps and enables evaluation of degree of dysplasia during ongoing endoscopy. This technology might offer considerable potential to ultimately fine-tune surveillance programs, particularly in high-risk groups.