The differentiation of homologous chromosomes as well as their parental origin can presently be conducted and determined exclusively by molecular genetic methods using microsatellite or SNP analysis. Only in exceptional cases is a distinction on a single-cell level possible, e.g. due to variations within the heterochromatic regions of chromosomes 1, 9, 16 and Y or the p-arms of the acrocentric chromosomes. In the absence of such polymorphisms, an individual distinction of the homologous chromosomes is not currently possible. Consequently, various questions of scientific and diagnostic relevance are unable to be answered. Based on the recently detected large-scale copy-number variations (LCV) or copy-number polymorphisms (CNP) spanning up to several megabase pairs of DNA, in this study, a molecular cytogenetic technique for the inter-individual differentiation of homologous chromosomes called parental-origin-determination fluorescence in situ hybridization (pod-FISH) is presented. All human chromosomes were covered with 225 LCV- and/or CNP-specific BAC probes, and one- to five-color chromosome-specific pod-FISH sets were created, evaluated and optimized. We demonstrated that pod-FISH is suitable for single-cell analysis of uniparental disomy (UDP) in clinical cases such as Prader-Willi syndrome caused by maternal UPD. A rare clinical case with a mosaic form of a genome-wide isodisomy was used to determine the detection limits of pod-FISH. Additionally we analyzed the informativeness of conventional microsatellite analysis for the first time and compared the results to pod-FISH. With this new possibility to study the parental origin of individual human chromosomes on a single-cell level, new doors for diagnostic and basic research are opened.