The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA (rDNA) of fish cestodes of the genus Eubothrium were sequenced. The ITS sequences of Eubothrium rugosum were determined and compared with previously analysed congeneric species, Eubothrium crassum and Eubothrium salvelini. The ITS-1 sequences of E. rugosum and E. crassum were 535 bp long, the length of E. salvelini ITS-1 was 536 bp. The ITS-2 region was found to be 403 bp in E. rugosum and E. crassum, and 401-402 bp in E. salvelini. The ITS-1 region of E. rugosum was closer to E. salvelini (identity 98.9-99.1%) than to E. crassum(97.9-98.1%), while the sequence similarity within the ITS-2 region was almost identical (97.5-98.0% for E. crassum; 97.7-98.3% for E. salvelini). Several restriction enzymes were found to be suitable for the differentiation of the three Eubothrium species by PCR-RFLP. The intraspecific sequence variation of E. crassum from different fish hosts (freshwater and marine) and European regions was very low, 0.2% for ITS-1 and 0.5% for ITS-2. Analysis of the ITS sequences of specimens from rainbow trout from three localities in Scotland revealed that both E. crassum and E. salvelini are present in this fish host.