BackgroundThe tobacco thrips (Frankliniella fuscaHinds; family Thripidae; order Thysanoptera) is an important pest that can transmit viruses such as the tomato spotted wilt orthotospovirus to numerous economically important agricultural row crops and vegetables. The structural and functional genomics within the order Thysanoptera has only begun to be explored. Within the > 7000 known thysanopteran species, the melon thrips (Thrips palmiKarny) and the western flower thrips (Frankliniella occidentalisPergrande) are the only two thysanopteran species with assembled genomes.ResultsA genome ofF. fuscawas assembled by long-read sequencing of DNA from an inbred line. The final assembly size was 370 Mb with a single copy ortholog completeness of ~ 99% with respect to Insecta. The annotated genome ofF. fuscawas compared with the genome of its congener,F. occidentalis. Results revealed many instances of lineage-specific differences in gene content. Analyses of sequence divergence between the twoFrankliniellaspecies’ genomes revealed substitution patterns consistent with positive selection in ~ 5% of the protein-coding genes with 1:1 orthologs. Further, gene content related to its pest status, such as xenobiotic detoxification and response to an ambisense-tripartite RNA virus (orthotospovirus) infection was compared withF. occidentalis. SeveralF. fuscagenes related to virus infection possessed signatures of positive selection. Estimation of CpG depletion, a mutational consequence of DNA methylation, revealed thatF. fuscagenes that were downregulated and alternatively spliced in response to virus infection were preferentially targeted by DNA methylation. As in many other insects, DNA methylation was enriched in exons inFrankliniella, but gene copies with homology to DNA methyltransferase 3 were numerous and fragmented. This phenomenon seems to be relatively unique to thrips among other insect groups.ConclusionsTheF. fuscagenome assembly provides an important resource for comparative genomic analyses of thysanopterans. This genomic foundation allows for insights into molecular evolution, gene regulation, and loci important to agricultural pest status.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09375-5.