Salmonid herpesvirus 1 (SalHV-1) is a pathogen of the rainbow trout (Oncorhynchus mykiss). Restriction endonuclease mapping, cosmid cloning, DNA hybridization, and targeted DNA sequencing experiments showed that the genome is 174.4 kbp in size, consisting of a long unique region (U(L); 133.4 kbp) linked to a short unique region (U(S); 25.6 kbp) which is flanked by an inverted repeat (R(S); 7.7 kbp). U(S) is present in virion DNA in either orientation, but U(L) is present in a single orientation. This structure is characteristic of the Varicellovirus genus of the subfamily Alphaherpesvirinae but has evidently evolved independently, since an analysis of randomly sampled DNA sequence data showed that SalHV-1 shares at least 18 genes with channel catfish virus (CCV), a fish herpesvirus whose complete sequence is known and which is unrelated to mammalian herpesviruses. The use of oligonucleotide probes demonstrated that in comparison with CCV, the conserved SalHV-1 genes are located in U(L) in at least five rearranged blocks. Large-scale gene rearrangements of this type are also characteristic of the three mammalian herpesvirus subfamilies. The junction between two SalHV-1 gene blocks was confirmed by sequencing a 4,245-bp region which contains the dUTPase gene, part of a putative spliced DNA polymerase gene, and one other complete gene. The implications of these findings in herpesvirus taxonomy are discussed.