Endogenous fatty acid synthesis is up-regulated in breast cancer. Phospholipids have been implicated in a number of cellular processes including signaling, proliferation, and apoptosis, but their role in cancer is largely unknown. In an effort to understand how lipids mediate tumor progression and oncogene expression, intact human various breast cancer cells having were interrogated by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). This in vitro cell-based workflow provides rapid and detailed analysis with little sample preparation compared to traditional lipid separation and extraction methods. Principal component analyses of cell lipid profiles allowed identification of cell lines based on differential expression of both Her2 and p53 genes, differences in the effect of these mutations on cancer progression. Breast cancer cell lines representing changes in metastatic potential and grade were also easily distinguished within a single assay. Tandem MS analyses identified of over 200 individual lipid species in negative ion mode, which is currently the most comprehensive phospholipid composition of breast cancer cells. These results demonstrate that DESI-MS based assays are powerful tools for rapidly classifying of cancer cells according to their geno- and phenotypes, while simultaneously providing extensive lipid compositions relevant to elucidating important biochemical mechanisms in cancer progression.