We report the molecular characterization of the amplification products obtained by specific PCR experiments aimed to identify the 5S ribosomal spacer of Setaria tundra specimens isolated from roe deer (Capreolus capreolus) in north Italy, which represent the first record of the parasite in this country. Three different fragments of approximately 400, 800, and 1200 bp (base pairs) are produced. Sequence analyses showed that all three fragments share a very high level of similarity to 5S spacer sequences of some Setaria species and other filariae present in genebank. Based on these sequences, we were able to design species-specific PCR primers for the precise identification of S. tundra.