Tumour heterogeneity leads to variable clinical response and inaccurate diagnostic and prognostic assessment. Cancer stem cells (CSCs) represent a subpopulation responsible for invasion, metastasis, therapeutic resistance, and recurrence in many human cancer types. However, the true identity of colorectal cancer (CRC) SCs remains elusive. Here, we aimed to characterize and define the gene expression portrait of CSCs in CRC-model SW403 cells. We found that ALDH⁺ positive cells are clonogenic and highly proliferative; their global gene expression profilingbased molecular signature revealed gene enrichment related to DNA damage, MAPK, FAK, oxidative stress response, and Wnt signalling. ALDH⁺ cells showed enhanced ROS stress resistance, whereas MAPK/FAK pathway pharmacologic inhibition limited their survival. Conversely, 5-fluorouracil increased the ALDH⁺ cell fraction among the SW403, HCT116 and SW620 CRC models. Notably, analysis of ALDH1A1 and POU5F1 expression levels in cohorts of 462 or 420 patients for overall (OS) or disease-free (DFS) survival, respectively, obtained from the Cancer Genome Atlas CRC dataset, revealed strong association between elevated expression and poor OS (p = 0.006) and poor DFS (p = 0.05), thus implicating ALDH1A1 and POU5F1 in CRC prognosis. Our data reveal distinct molecular signature of ALDH⁺ CSCs in CRC and suggest pathways relevant for successful targeted therapies and management of CRC.