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The bovine lungworm Dictyocaulus viviparus is found in cattle and deer species (roe deer, fallow deer). Although a separate lungworm species (D. eckerti) from roe deer has been described, this description, based on minor morphological differences, is not generally accepted. The aim of the present study was to examine genetic differences among four lungworm species isolated from cattle, sheep, horses, and fallow deer. Ribosomal second internal transcribed spacers (ITS2) of individual worms of all species were amplified by polymerase chain reaction (PCR), and restriction-fragment-length polymorphisms were analyzed after digestion with five different restriction enzymes. After electrophoretic separation of the digested PCR products, all four lungworm species showed different banding patterns. Lungworms from cattle (D. viviparus) and those from fallow deer (D. eckerti) were genetically distinct. These results are consistent with the hypothesis that the taxonomic classification of D. eckerti as a separate species is correct.