Simple SummarySpongy moth, formerly known as European gypsy moth, is an invasive forest pest in the United States and is under quarantine in the eastern part of the country. The flighted spongy moth complex, a group of related spongy moths formerly known as Asian gypsy moth, are pests that are not currently present in the United States. As part of our research to characterize spongy moth pathogens, we determined the genome sequences and relative amounts of genomes of an iflavirus, an RNA-based virus, from spongy moths in two locations in the USA and flighted spongy moths in two locations in China. The iflaviruses from the USA and China spongy moth populations were distinguishable from each other on the basis of their genome sequences. Iflavirus genome levels in all moth samples were very high, but these high genome levels were not associated with any obvious disease symptoms. The information in this study raises questions about the effect of RNA viruses such as iflaviruses on their hosts, and will be of interest to scientists who work with iflaviruses, spongy moth, and other forest pests.AbstractThe spongy moth virus Lymantria dispar iflavirus 1 (LdIV1), originally identified from aLymantria disparcell line, was detected in 24 RNA samples from female moths of four populations from the USA and China. Genome-length contigs were assembled for each population and compared with the reference genomes of the first reported LdIV1 genome (Ames strain) and two LdIV1 sequences available in GenBank originating from Novosibirsk, the Russian Federation. A whole-genome phylogeny was generated for these sequences, indicating that LdIV1 viruses observed in North American (flightless) and Asian (flighted) spongy moth lineages indeed partition into clades as would be expected per their host’s geographic origin and biotype. A comprehensive listing of synonymous and non-synonymous mutations, as well as indels, among the polyprotein coding sequences of these seven LdIV1 variants was compiled and a codon-level phylogram was computed using polyprotein sequences of these, and 50 additional iflaviruses placed LdIV1 in a large clade consisting mostly of iflaviruses from other species of Lepidoptera. Of special note, LdIV1 RNA was present at very high levels in all samples, with LdIV1 reads accounting for a mean average of 36.41% (ranging from 1.84% to 68.75%, with a standard deviation of 20.91) of the total sequenced volume.