We describe a dataset obtained by applying our recently developed iFISH pipeline to simultaneously visualize 16 small (size range: 62–73 kilobases, kb) evenly spaced DNA loci on chromosome 2 in human cells, in a single round of hybridization. We show how, using combinatorial color coding, multiple loci can be precisely 3D localized in single cells, and how inter-loci distances scale inversely with chromosome contact frequencies determined by Hi-C, as expected. We provide raw images and...