Several different techniques have been employed to detect and identify Ichthyophonus spp. in infected fish hosts; these include macroscopic observation, microscopic examination of tissue squashes, histological evaluation, in vitro culture, and molecular techniques. Examination of the peer-reviewed literature revealed that when more than 1 diagnostic method is used, they often result in significantly different results; for example, when in vitro culture was used to identify infected trout in an experimentally exposed population, 98.7% of infected trout were detected, but when standard histology was used to confirm known infected tissues from wild salmon, it detected ∼50% of low-intensity infections and ∼85% of high-intensity infections. Other studies on different species reported similar differences. When we examined a possible mechanism to explain the disparity between different diagnostic techniques, we observed non-random distribution of the parasite in 3-dimensionally visualized tissue sections from infected hosts, thus providing a possible explanation for the different sensitivities of commonly used diagnostic techniques. Based on experimental evidence and a review of the peer-reviewed literature, we have concluded that in vitro culture is currently the most accurate diagnostic technique for determining infection prevalence of Ichthyophonus, particularly when the exposure history of the population is not known.