With the progress of array technologies and the enabled screening of individual human genomes, a new kind of polymorphism has been described - the so-called copy number variation (CNV) polymorphism. Copy number variants can be found in around 12% of the human genome sequence and have a size of up to several hundred kilobase pairs. These variants can not only differ between individuals, but also between corresponding alleles on homologous chromosomes. We recently developed a cytological assay for parental origin determination that relies on the design of CNV-based sets of probes for fluorescence in situ hybridization (POD-FISH). Here we describe an improved POD-FISH protocol that exploits "high frequency" variants for better discrimination of homologous chromosomes.