Here, we present FishPIE, a nested PCR primer set of 82 phylogenetic informative exon markers for ray-finned fish phylogenetic analysis. By testing 203 species from 31 orders of Actinopterygia, we showed that the primers exhibit high PCR success rate (~75%). To examine the phylogenetic utility of the FishPIE markers in resolving phylogenetic relationships at different taxonomic levels, we combined orthologous sequences of the FishPIE markers from publicly available fish genomes and transcriptomes and constructed the phylogeny of totally 754 specimens from 710 fish species belonging to 190 families and 60 orders.