With the current trend for early breast cancer detection, there is also a growing demand for discovering markers to assist in the patient risk stratification. Molecular chaperones play essential roles in the post-translational maturation of oncogenic client proteins and are strongly associated with carcinogenesis. To better define the role of chaperones in breast cancer, tissue arrays were immunostained for the chaperones Hsp90 and p23 and assessed in terms of reactivity, intensity and cellular localization. Cytoplasmic Hsp90 protein expression was significantly stronger in ductal carcinoma in situ and invasive breast carcinomas as compared to normal breast tissue (p < 0.0001). Importantly, invasive cores also display a significant relationship between nuclear Hsp90 expression and TNM stage (p = 0.0023) as well as estrogen receptor (ER) status (p = 0.0112). Expression of p23 was substantially stronger in normal tissue than in malignant carcinomas (p < 0.0001) with no discernable correlation with TNM stage, which differs from in vitro results. To determine the functional differences between normal and tumor cells, we assessed the bound versus free forms of Hsp90. Contrary to previous results, we find both complexed and free Hsp90 in normal, immortal and tumor cells, suggesting that the differences between normal and immortal/cancer cells in terms of functional Hsp90 levels may be related to either its overall expression level or its subcellular location. Taken together, our data suggest that blocking nuclear Hsp90 function in advanced breast cancers may be a more targeted approach to treating advanced breast cancers that are refractory to traditional therapy.