摘要:SummaryUnderstanding the evolutionary history of the highly diverse ray-finned fishes has been challenging, and the development of more universal primers for phylogenetic analyses may help overcoming these challenges. We developed FishPIE, a nested PCR primer set of 82 phylogenetically informative exon markers, and tested it on 203 species from 31 orders of Actinopterygii. We combined orthologous sequences of the FishPIE markers obtained from published genomes and transcriptomes and constructed the phylogeny of 710 species belonging to 190 families and 60 orders. The resulting phylogenies had topologies comparable to previous phylogenomic studies. We demonstrated that the FishPIE markers could address phylogenetic questions across broad taxonomic levels. By incorporating the newly sequenced taxa, we were able to shed new light on the phylogeny of the highly diverse Cypriniformes. Thus, FishPIE holds great promise for generating genetic data for broad taxonomic groups and accelerating our understanding of the fish tree of life.Graphical abstractDisplay OmittedHighlights•FishPIE is a nested PCR primer set of 82 markers for fish phylogenetic analysis•The markers can be broadly applied to all orders of ray-finned fishes•Their phylogenetic performance is comparable to that of genomic analysesEvolutionary biology; Evolutionary processes; Phylogenetics