HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocalization analyses of the endogenous MEC1-HS1, indicating an increased interaction with Vimentin at the cell adhesion sites. In CLL cells isolated from patients with poor prognosis, we observed a larger accumulation of HS1 at the basal region and a higher density of HS1 nanoclusters in the central regions of the cells if compared to good-prognosis CLL and healthy B cells, suggesting a different role for the protein in the cell types analyzed. Our 3D-STED approach lays the ground for revealing tiny differences of HS1 distribution, its functionally active forms, and colocalization with protein partners.