In order to explore the value of p63, smoothmuscle actin (alpha-SMA) and cytokeratin 5/6 (CK5/6) in the differential diagnosis of ductal lesions of breast, 88 tissue specimens of ductal lesions of breast were collected and examined histologically by HE staining. By using immunohistochemistry, the expression of p63, alpha-SMA and CK5/6 was detected. The results showed that in 38 cases of benign breast lesions, the proliferating cells were all positive for p63 and alpha-SMA. In 19 cases of ductal carcinoma in situ (DCIS) and 7 cases of intraductal papillary carcinoma, alpha-SMA positive cells formed a layer of continuous embroider-shaped structure and the p63 positive cells formed a layer of evenly separated embroider-shaped structure around the ducts. There was no cross-reaction between p63 and interstitial myofibroblasts and vascular smooth muscle cells. In 38 cases of benign breast lesions, the positive rate of CK5/6 expression was 100%. In 5 cases of atypical ductal hyperplasia, there were few positive cells in the ducts. In 19 cases of CDIS, no tumor cells expressed CK5/6. In 19 cases of invasive ductal carcinoma, almost no CK5/6 was detectable. It was suggested that p63 could serve as a novel specific marker for the identification of breast myoepithelial cells. CK5/6 is of value in differentiating ductal proliferation of varying degrees, especially in the differentiation between cancerous and non-cancerous changes. Simultaneous detection of p63, CK5/6 and alpha-SMA can help increase the diagnostic accuracy of breast diseases.