The global clustering approach provided high-quality imaging, often displaying sub-micrometric spatial features consistent with those described in classification guidelines (e.g., variation of nuclear-to-cytoplasmic ratios among different subtypes, localization of specific biomarkers (MPO)) or suggesting new possible subtype-specific features (e.g., carotenoids)). Moreover, this approach enabled a semiquantitative and objective characterization of a substantial amount of hyperspectral data. Visual interpretation (images and bar plots) and multivariate analysis (LDA) unveiled maturation and differentiation trends across various leukemia subtypes, particularly highlighted by MPO, carotenoids, and HB expression. Encouraging results were observed in classification, especially for AML M0, AML M6, and AML M5a, as well as when grouping major leukemia categories. To the best of our knowledge, this study represents the most extended Raman characterization of leukemia subtypes from patients but also the most extended spontaneous Raman imaging study of single cells from patients, reporting images from >300 cells, analyzed using a single clustering analysis step.