In order to explore the value of p63, smooth muscle actin (α-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, α-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 α-SMA. In 19 cases of ductal carcinoma in situ (DCIS) and 7 cases of intraductal papillary carcinoma, α-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 α-SMA can help increase the diagnostic accuracy of breast diseases.