Fine needle aspiration cytology is central to the evaluation of clinically or mammographically detected suspicious lesions of the breast. On the basis of results from studies of >500 breast cancers by comparative genomic hybridization we have developed protocols and designed probe sets that allow one to visualize recurrent chromosomal aneuploidies, amplification of oncogenes, and deletion of tumor suppressor genes directly in cytological preparations using multicolor fluorescence in situ hybridization. The fluorescence in situ hybridization probes are specific for chromosome arm 1q, the c-MYC and HER2 oncogenes, the tumor suppressor gene p53 and, as controls for chromosome ploidy of each cell, the centromeres of chromosomes 8, 10, and 17. Application of these diagnostic mixtures to 20 invasive breast cancers, 7 mastopathias, and 2 fibroadenomas demonstrates that a highly sensitive, specific, and objective diagnosis of breast cancer is now possible on cytological preparations obtained by minimally invasive fine needle aspiration.