Breast cancer tumors are composed of heterogeneous cell populations. These populations display a high variance in morphology, growth and metastatic propensity. They respond differently to therapeutic interventions, and some may be more prone to cause recurrence. Studying individual subpopulations of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments on cryostat sections of human breast cancer tissue. This method allows for selection of recombinant antibodies binding to antigens specifically expressed in a small part of the tissue section. In this case, a CD271+ subpopulation of breast cancer cells was targeted, and these may be potential breast cancer stem cells. We isolated an antibody fragment LH 7, which in immunohistochemistry experiments demonstrates specific binding to breast cancer subpopulations. The selection of antibody fragments directly on small defined areas within a larger section of malignant tissue is a novel approach by which it is possible to better target cellular heterogeneity in proteomic studies. The identification of novel biomarkers is relevant for our understanding and intervention in human diseases. The selection of the breast cancer-specific antibody fragment LH 7 may reveal novel subpopulation-specific biomarkers, which has the potential to provide new insight and treatment strategies for breast cancer.