Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode‐resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross‐hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species‐specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species‐specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. 1 Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta.