The genus Populus is one of the important tree species in Korean peninsula and many other countries in the world. It represents the model species of forest genomics because it grows fast and reproduces rapidly. In this reason, their genetic characteristics have been well studied and the whole genome has been sequenced completely in some species. However, cytogenetic study of the genus Populus has been limited. In the present study, karyotypes of Korean aspen (P. tremula var. davidiana), Silver poplar (P. alba) and their two hybrids, Suwon aspen (P. tremula var. glandulosa) and Hyun aspen (P. alba × P. tremula var. glandulsa) were analyzed by means of the fluorescence in situ hybridization (FISH). Root samples were collected from mature trees in the demonstration forest, located at Suwon, Kyonggi province in South Korea. The fresh root cells were examined by DAPI (4',6-diamidino-2-phenylindole) staining and FISH using 45S rDNA and 5S rDNA probes. As the results, the chromosome compositions of all species were the same as 2n = 38. The karyotype formulas of Korean aspen, Silver poplar, Suwon aspen and Hyun aspen were 28m + 6sm + 4st (2sat), 26m + 10sm (2sat) + 2st, 26m + 12sm (2sat) and 28m + 10sm (2sat), respectively. The four species had one pair of 45S rDNA site and one pair of 5S rDNA site in common with FISH karyotypes. The similarity of FISH karyotypes among four species indicated close genetic relationship and coexistence of their interspecific hybrids. This research will provide genetic information on cytogenetic research of Populus and genetic mapping that can be applied to the breeding program of Populus in the near future.