Abstract Background The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer predicts poor clinical outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) features display enhanced malignant and metastatic potential. A new methodology was developed in order to investigate the co-expression of a stemness and an EMT marker (ALDH1 and TWIST, respectively) on single CTCs of patients with early and metastatic breast cancer. Methods Triple immunofluorescence using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies was performed in cytospins prepared from hepatocellular carcinoma HepG2 cells and SKBR-3, MCF-7 and MDA.MB.231 breast cancer cell lines. Evaluation of ALDH1 expression levels (high, low or absent) and TWIST subcellular localization (nuclear, cytoplasmic or absent) was performed using the ARIOL system. Cytospins prepared from peripheral blood of patients with early (n = 80) and metastatic (n = 50) breast cancer were analyzed for CTC detection (based on pan-cytokeratin expression and cytomorphological criteria) and characterized according to ALDH1 and TWIST. Results CTCs were detected in 13 (16%) and 25 (50%) patients with early and metastatic disease, respectively. High ALDH1 expression (ALDH1 high ) and nuclear TWIST localization (TWIST nuc ) on CTCs was confirmed in more patients with metastatic than early breast cancer (80% vs. 30.8%, respectively; p = 0.009). In early disease, ALDH1 low/neg CTCs (p = 0.006) and TWIST cyt/neg CTCs (p = 0.040) were mainly observed. Regarding co-expression of these markers, ALDH1 high /TWIST nuc CTCs were more frequently evident in the metastatic setting (76% vs. 15.4% of patients, p = 0.001; 61.5% vs. 12.9% of total CTCs), whereas in early disease ALDH1 low/neg /TWIST cyt/neg CTCs were mainly detected (61.5% vs. 20% of patients, p = 0.078; 41.9% vs. 7.7% of total CTCs). Conclusions A new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the single CTC-level in patients with breast cancer. A differential expression pattern for these markers was observed both in early and metastatic disease. CTCs expressing high ALDH1, along with nuclear TWIST were more frequently detected in patients with metastatic breast cancer, suggesting that these cells may prevail during disease progression.