Digenean eye-flukes of the genus <i>Diplostomum </i>(Family:  Diplostomatidae) are common in the eyes of freshwater fishes.  <i>Diplostomum </i>spp. infections can lead to severe detrimental deformities and even death to both farmed and wild fish species.  Identification of <i>Diplostomum</i> spp. in the fish host (metacercaria) is challenging because species are morphologically indistinguishable.  Since <i>Diplostomum</i> spp. are difficult to identify the literature is confounded by synonymy and as a result, the taxonomic status of the genus remains unclear.  Thus, it is difficult to ascertain the pathological status, diversity and geographical distribution of this economically important group of parasites. The purpose of this study was to define operational taxonomic units (OTU) using morphometric and molecular data collected from <i>Diplostomum </i>spp. metacercaria in the U.K.  Data was collected from ten fish species and four host locations:  lens humor retina and brain.  Four OTU were defined by host location using morphometric analysis.  Hence the morphometric analysis proved metacercaria from the four host locations were different in size and shape. Four OTU were discriminated using PCR-RFLP and sequence analysis.  However, with the exception of brain parasites, those in the fish eye (lens, humor and retina) could not be differentiated by host location.  Therefore, although parasites in the three fish eye locations were different in size and shape, they were not genetically different.  The OTU ITS1 sequence data was compared to published <i>Diplostomum</i> spp. sequences and the following genetic similarity was revealed:  OTU1 showed 99.4% similarity to <i>D. mergi</i>;  OTU2 99.7% similarity to <i>D. baeri</i> and OTU3 99.7% similarity to <i>D. paracaudum/D. spathaceum.  </i>OTU4 in the fish brain has previously been described as <i>D. phoxini.</i> The molecular approach was subsequently used in a temporal study to assess <i>Diplostomum </i>spp. metacercaria infections over the course of a year in a rainbow trout farm.