Interphase multicolor fluorescence in situ hybridization (IM-FISH) has great promise for improving cancer diagnosis because it can directly visualize multiple changes in chromosomes and gene copy number on a cell-to-cell basis. However, no more than four targets can be detected simultaneously by current commercially available IM-FISH protocols, and the DNA probes used are too large to detect the single-gene aberrations that characterize tumorigenesis. As a result, multiple FISH has a low sensitivity in detecting cancer cells. To overcome such limitations, we first developed specific genomic probes for the genes relevant to primary lung cancer. We next designed a multiple FISH array by arranging four different compositions of cocktails of four probes for each gene on a coverslip, which allowed four four-color FISH experiments to be performed in parallel on a single slide. We then tested the multiple FISH array on bronchial brushing samples from lung cancer patients to determine its ability to detect genetic abnormalities. A comparison of the data with the results of cytology and commercial four-color FISH suggested that the multiple FISH array had the highest sensitivity for cancer detection. The technique may thus be a powerful laboratory strategy for cancer prevention and early detection and for improved patient management.