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Simple Summary: Detection of colorectal carcinoma is performed visually by investigators and is confirmed pathologically. With hyperspectral imaging, an expanded spectral range of optical information is now available for analysis. The acquired recordings were analyzed with a neural network, and it was possible to differentiate tumor from healthy mucosa in colorectal carcinoma by automatic classification with high reliability. Classification and visualization were performed based on a four-layer perceptron neural network. Based on a neural network, the classification of CA or AD resulted in a sensitivity of 86% and a specificity of 95%, by means of leave-one-patient-out cross-validation. Additionally, significant differences in terms of perfusion parameters (e.g., oxygen saturation) related to tumor staging and neoadjuvant therapy were observed. This is a step towards optical biopsy. Currently, colorectal cancer (CRC) is mainly identified via a visual assessment during colonoscopy, increasingly used artificial intelligence algorithms, or surgery. Subsequently, CRC is confirmed through a histopathological examination by a pathologist. Hyperspectral imaging (HSI), a non-invasive optical imaging technology, has shown promising results in the medical field. In the current study, we combined HSI with several artificial intelligence algorithms to discriminate CRC. Between July 2019 and May 2020, 54 consecutive patients undergoing colorectal resections for CRC were included. The tumor was imaged from the mucosal side with a hyperspectral camera. The image annotations were classified into three groups (cancer, CA; adenomatous margin around the central tumor, AD; and healthy mucosa, HM). Classification and visualization were performed based on a four-layer perceptron neural network. Based on a neural network, the classification of CA or AD resulted in a sensitivity of 86% and a specificity of 95%, by means of leave-one-patient-out cross-validation. Additionally, significant differences in terms of perfusion parameters (e.g., oxygen saturation) related to tumor staging and neoadjuvant therapy were observed. Hyperspectral imaging combined with automatic classification can be used to differentiate between CRC and healthy mucosa. Additionally, the biological changes induced by chemotherapy to the tissue are detectable with HSI. [ABSTRACT FROM AUTHOR]