Except individuals with variations in the pericentric heterochromatic chromosome regions (including acrocentric short arms), it is not possible to distinguish homologous chromosomes at the single-cell level. Due to this limitation, various questions of scientific and diagnostic relevance could not be investigated until now. Based on copy number variations (CNV) of up to several megabase pairs of DNA, we have developed a molecular cytogenetic approach for the interindividual differentiation of homologous chromosomes, the so-called parental origin determination FISH (pod-FISH) technique. To this end, all human chromosomes were covered with CNV-comprehensive BAC probes in one- to five-color chromosome-specific pod-FISH sets. This approach to investigating the parental origin of individual human chromosomes at the single-cell level has opened up new horizons for diagnostics and basic research.