The V4 locus within the 18S ribosomal gene is relatively variable but surrounded by highly invariant regions. This allowed Cunningham et al. (1995) to design primers in the absence of Gyrodactylus-specific sequence information and successfully amplify the region. Based on Cunningham's primers, V4 has been amplified in a number of gyrodactylids from salmonid hosts, and has proved useful in discriminating most using restriction fragment polymorphisms.

Cunningham's primers for V4 locus: Amplification conditions: