The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 μm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as “distinct base region” spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations could point to a cancer SC phenotype; an approach capable of identifying their in situ location has enormous therapeutic applications.