Primary acantholytic squamous cell carcinoma (ASCC) of the breast is a rare and aggressive variant of invasive breast cancer. Here we report two new cases of ASCC and their immunohistochemical and cytogenetic characterization. One case was associated with systemic metastases and death and the other with local failure prior to loss of follow-up. Using comparative genomic hybridization (CGH), both tumors showed a high overall number of chromosomal imbalances with a similar pattern of gains and losses. Genetic aberrations common to both tumors included losses at 3p11-p25, 5q21-q31, 8p, 9, 13p13-q21, 16q12-q21, and 17p and gains at 1q31-qter, 7p, 18q12-qter, 19q, and 20. Immunohistochemically, the tumors were characterized by high proliferative activity, an uncommon cytokeratin expression profile, reduced E-cadherin staining, and overexpression of p53 and epithelial growth factor receptor (EGFR). The results of our analyses suggest that genetic alterations observed in ASCC of the breast include imbalances commonly observed in both mammary adenocarcinoma and squamous cell carcinoma of other locations. Furthermore, the overexpressed EGFR could be a possible therapeutic target for individual cases of this aggressive tumor type.