DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitates analysis of the FISH results with the FISH-FINDER computer program.