Dactylogyrus spp. are monogenean worms found mostly as ectoparasites on the gills of several fish species, including carp and goldfish. These parasites are commonly detected by microscopic analysis of the gill lamellae, but this is time-consuming and technically difficult. In contrast to this conventional method, molecular techniques provide specific, sensitive and safe detection of parasites. In the present study, polymerase chain reaction (PCR) and subsequent DNA sequencing were used to detect Dactylogyrus spp. Specific common primers were designed to amplify the ITS-1 region of the rRNA gene of Dactylogyrus spp. Dactylogyrus worms were collected from 100 goldfishes and identified using a dissection microscope. Then, single worms were used for DNA extraction. To evaluate the PCR, a single parasite was added to a parasite-free gill, which then had its DNA extracted. Subsequently, the PCR products were purified and sequenced. Comparison of the nucleotide sequences of the PCR products with GenBank sequences showed that there was 100% homology with sequences from two Dactylogyrus spp., namely Dactylogyrus vastator and Dactylogyrus dulkeiti (registered under accession numbers AJ 564159 and AJ 564126, respectively). The results obtained from sequence analyses were consistent with species identification by microscopy. Therefore, the results show that it is possible to develop a sensitive and precise PCR method for the detection of Dactylogyrus-infected fish using DNA extracted from the whole gill.