Purpose To compare circulating tumor cell (CTC) detection rates in patients with early (M₀) and metastatic (M₊) breast cancer using 2 positive-selection methods or size-based unbiased enrichment. Methods Blood collected at baseline and at different times during treatment from M₀ patients undergoing neoadjuvant therapy and from M₊ women starting a new line of treatment was processed in parallel using AdnaTest EMT-1/ and EMT-2/Stem CellSelect/Detect kits or ScreenCell Cyto devices. CTC positivity was defined according to the suggested cutoffs and cytological parameters, respectively. Results Higher CTC detection rates were obtained with the AdnaTest approach when using for CTC-enrichment antibodies against ERBB2 and EGFR in addition to MUC1 and the classical epithelial surface marker EPCAM (13% vs. 48%). In M₀ patients mainly, CTC positivity rates further increased when EMT- and stemness-related marker expression (PIK3CA, AKT2 and ALDH1) was evaluated in addition to EPCAM, MUC1 and ERBB2. When the physical properties of tumor cells were exploited, CTCs were detected at higher percentages than with positive-selection-based methods, without any difference between clinical stages (78% in M₀ vs. 72% in M₊ cases at baseline). Circulating tumor microemboli (CTMs) were detected in addition to single CTCs with significantly higher frequency in M₀ than M₊ samples (78% vs. 27%, p = 0.0002). Conclusions Different approaches for CTC detection probably identify distinct tumor cell subpopulations, but need technical standardization before their clinical validity and biological specificity may be adequately investigated. The distinct role of CTMs compared with CTCs as prognostic and predictive biomarkers represents a further challenge.