Molecular characterization of breast cancer is pivotal for identifying new molecular targets and determining the appropriate treatment choices. Advances in molecular profiling technology have given greater insight into this heterogeneous disease, over and above hormone receptor and human epidermal growth factor receptor 2 status. Agents targeting recently characterized molecular biomarkers are under clinical development; the success of these targeted agents is likely to depend on identifying the patient population most likely to benefit. Therefore, clinical trials of breast cancer often require prescreening for, or stratification by, relevant molecular markers or exploratory analyses of biomarkers that can predict or monitor the response to treatment. Consequently, the role of the pathologist has become increasingly important. The key considerations for pathologists include tissue availability, ownership of archival tissue, type of diagnostic/biomarker test required, method of sample processing, concordance between different tests and testing centers, and tumor heterogeneity. In the present review, we explore how pathology is used in current clinical trials of breast cancer and describe the various technologies available for molecular testing. Furthermore, the factors required for the successful application of pathology in clinical trials of breast cancer and the issues that can arise and how these can be circumvented are discussed.