As a result of breast cancer screening programs, high-grade ductal carcinoma in situ (DCIS) of the breast is diagnosed more often. Frequently, a DCIS diagnosis can only be made using immunohistochemical stains to visualize the myoepithelial layer in order to assess microinvasion. Standard markers for myoepithelial cells are CK5/6 and p63. An isoform of the latter, ∆Np63, is recognized by a recently developed antibody, p40. Here, we compare the standard myoepithelial markers CK5/6 and p63 with p40. We immunostained full sections of tissue samples of 35 high-grade DCIS and compared the staining pattern of CK5/6, p63 and p40 in tumour tissue and in normal glands. Staining patterns of myoepithelial cells for p63 and p40 were similar in terms of the percentage of stained nuclei. In all cases, p63 was strongly expressed, while this was the case for p40 in 31 (89%) and moderately in 4 (11%) cases. All but one case (97%) showed a similar percentage of stained myoepithelial cells in comparing CK5/6 and p40 staining. CK5/6 expression was heterogeneous and strong/moderate/weak in 60, 34 and 6 % respectively. Compared to surrounding normal glands, staining of myoepithelial cells for all three markers in the neoplastic lesion was attenuated. In high-grade DCIS, p40 staining is highly specific for myoepithelial cells. Its staining pattern and intensity are equal to p63, which opens up its use for daily practice. Staining with p40 is less heterogeneous than that for CK5/6.