Since its discovery by Koch in 1989, primed in situ labeling (PRINS) reaction provides an alternative approach for direct detection of human chromosomes. The multiple color (multi)-PRINS technique can simultaneously and specifically display different chromosomes with different colors in the same metaphase or interphase nucleus by using sequential labeling of different chromosome targets. We developed a triple-PRINS reaction on uncultured amniotic cells by omitting the blocking step and taking advantage of mixing two fluorochromes (fluorescein and rhodamine) to create a third color for simultaneous detection in the same amniocytes of three different chromosome targets, e.g., chromosomes 18, X, and Y. Fluorescent signals corresponding to chromosomes 18, X, and Y were shown as yellow, red, and green color spots, respectively. Multi-PRINS is as accurate and reliable as multicolor fluorescent in situ hybridization (multi-FISH) for the detection of aneuploidies involving chromosomes 18, X, and Y. Furthermore, multi-PRINS represents a faster and more cost-effective alternative to FISH for prenatal testing of aneuploidy in uncultured amniocytes.