We examined host specificity of a recently described parasite (Gyrodactylus tularosae Kritsky and Stockwell, 2005) to its natural host, the White Sands pupfish (Cyprinodon tularosa Miller and Echelle, 1975), compared with a closely related congener, the sheepshead minnow (Cyprinodon variegatus). In each of 8 replicates, 1 uninfected C. tularosa and 1 uninfected C. variegatus were exposed to 3 C. tularosa infected with G. tularosae over a 4-d exposure trial. Focal fish were subsequently isolated for 5 d to evaluate infection persistence. Experiment-wide fluke infection prevalence was 100% for C. tularosa throughout the experiment. Prevalence on C. variegatus increased to 100% by d 3 of exposure but declined in isolation to 50% on the last day of the experiment. Fluke intensity was significantly higher for C. tularosa than C. variegatus throughout the experiment. Following isolation, parasite intensity declined for both species. These data suggest that G. tularosae prefers its native host, C. tularosa, but it may be able to use C. variegatus as a transient host.