The aim of the current study was to assess the expression levels of c-Src and phosphorylated Src kinase in human breast cancers and to establish if these are linked to oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status or patient survival. Tissue microarray technology was used to analyze 314 breast cancer specimens. Immunohistochemistry was performed using antibodies to c-Src, Y419Src, and Y215Src, and expression was assessed using the weighted histoscore method. High cytoplasmic c-Src kinase and high membrane phosphorylated activated Y419Src kinase was associated with decreased disease-specific survival. In contrast, phosphorylated activated nuclear and cytoplasmic Y215Src kinase expression levels were significantly associated with improved disease-specific survival. When the cohort was subdivided according to ER/PR/HER2 status, the ER-negative subgroup (105 patients) was associated with improved disease-specific survival and was found to be independent by multivariate analysis with a hazard ratio of 0.4 (interquartile range 0.2–0.8). High cytoplasmic c-Src expression was associated with decreased survival; high expression of activated c-Src (Y215) was associated with improved survival. This was potentiated in the ER/HER2-negative subgroup. Hence, administration of Src kinase inhibitors aiming to decrease phosphorylation should be approached with caution, especially in ER-negative patients. It is therefore essential to appropriately identify with the correct biomarkers which patients are most likely to respond to Src inhibitors.