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5-aminolevulinic acid (5-ALA) introduction in the surgical management of Glioblastoma (GBM) enables the intra-operatively identification of cancer cells in the mass by means of fluorescence. Here, we analyzed the phenotype of GBM cells isolated from distinct tumour areas determined by 5-ALA (tumour core, 5-ALA intense and vague layers) and the potency of 5-ALA labelling in identifying GBM cells and cancer stem cells (CSCs) in the mass. 5-ALA identified distinct layers in the mass, with less differentiated cells residing in the core of the tumour. 5-ALA was able to stain up to 68.5% of CD133(+) cells in the 5-ALA intense layer and, although 5-ALA(+) cells retrieved from different tumour areas contained a similar proportion of CD133(+) cells (range 27.5-35.6%), those from the vague layer displayed the lowest ability to self-renew. In conclusion, our data demonstrate that a substantial amount of GBM cells and CSCs in the mass are able to avoid 5-ALA labelling and support the presence of heterogenic CSC populations in the GBM mass.