Acute lymphoblastic leukemia (ALL) is the most common type of malignant neoplasms in the pediatric population. B-cell precursor ALLs (BCP-ALLs) are derived from the progenitors of B lymphocytes. Traditionally, risk factors stratifying therapy in ALL patients included age at diagnosis, initial leukocytosis, and the response to chemotherapy. Currently, treatment intensity is modified according to the presence of specific gene alterations in the leukemic genome. Raman imaging is a promising diagnostic tool, which enables the molecular characterization of cells and differentiation of subtypes of leukemia in clinical samples. This study aimed to characterize and distinguish cells isolated from the bone marrow of patients suffering from three subtypes of BCP-ALL, defined by gene rearrangements, i.e., BCR-ABL1 (Philadelphia-positive, t(9;22)), TEL-AML1 (t(12;21)) and TCF3-PBX1 (t(1;19)), using single-cell Raman imaging combined with multivariate statistical analysis. Spectra collected from clinical samples were compared with single-cell spectra of B-cells collected from healthy donors, constituting the control group. We demonstrated that Raman spectra of normal B cells strongly differ from spectra of their malignant counterparts, especially in the intensity of bands, which can be assigned to nucleic acids. We also showed that the identification of leukemia subtypes could be automated with the use of chemometric methods. Results prove the clinical suitability of Raman imaging for the identification of spectroscopic markers characterizing leukemia cells.