Ulva prolifera has been identified as the dominant species of the world’s largest green tides occurring in the Yellow Sea, China, using the molecular markers of internal transcribed spacer (ITS) and 5S ribosomal DNA (5S rDNA). In this study, these two markers were successfully located on the chromosomes of U. prolifera using the fluorescence in situ hybridization (FISH) technique. The ITS rDNA probe was localized on chromosome number 1 of gametophytes and the 5S rDNA probe appeared on chromosome number 6. Dual-colour FISH was also performed, and the locations of the two markers were consistent with single-colour localization, indicating a precise gene location. The localization of these rDNAs using the FISH technique facilitated the identification of individual chromosomes and a karyotype analysis of Ulva, which is beneficial for understanding the blooming mechanism of green tides in the Yellow Sea.