The presence of tumor-initiating cells (CD44⁺/CD24⁻) in solid tumors has been reported as a possible cause of cancer metastasis and treatment failure. Nevertheless, little is know about the presence of CD44⁺/CD24⁻ cells within the primary tumor and metastasis. The proportion of CD44⁺/CD24⁻ cells was analyzed in 40 samples and in 10 lymph node metastases using flow cytometry phenotyping. Anti-human CD326 (EpCam; FITC), anti-human CD227 (MUC-1; FITC), anti-human CD44 (APC), and anti-human CD24 (PE), anti-ABCG2 (PE), and anti-CXCR4 (PeCy7) were used for phenotype analysis. The mean patient age was 60.5 years (range, 33–87 years); mean primary tumor size (pT) was 1.8 cm (0.5–3.5 cm). The Wilcoxon or Kruskal–Wallis test was used for univariate analyses. Logistic regression was used for multivariate analysis. The median percentage of CD44⁺/CD24⁻ cells within primary invasive ductal carcinomas (IDC) was 2.7% (range, 0.2–71.2). In lymph node metastases, we observed a mean of 6.1% (range, 0.07–53.7). The percentage of CD44⁺/CD24⁻ cells in IDCs was not associated with age, pT, tumor grade and HER2. We observed a significantly enrichment of CD44⁺/CD24⁻ and ABCG2⁺ cells in ESA⁺ cell population in patients with positive lymph nodes (P = 0.02 and P = 0.04, respectively). Our data suggest that metastatic dissemination is associated with an increase in tumor-initiating cells in stage I and II breast cancer.