Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities. This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon. For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures. Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses. For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized. Tissues were collected 15-20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (approximately 20 g) showing severe gross and microscopic lesions, consistent with ISAV infection. Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish. Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.