Sex-specific ecology has management implications, but rapid sex-chromosome turnover in fishes hinders sex-marker development for monomorphic species. We used annotated genomes and reduced-representation sequencing data for two Australian percichthyids, Macquarie perch Macquaria australasica and golden perch M. ambigua, and whole genome resequencing for 50 Macquarie perch of each sex, to identify sex-linked loci and develop an affordable sexing assay. In silico pool-seq tests of 1,492,004 Macquarie perch SNPs revealed that a 275-kb scaffold was enriched for gametologous loci. Within this scaffold, 22 loci were sex-linked in a predominantly XY system, with females being homozygous for the X-linked allele at all 22, and males having the Y-linked allele at >7. Seven XY-gametologous loci (all males, but no females, are heterozygous or homozygous for the male-specific allele) were within a 146-bp region. A PCR-RFLP sexing assay targeting one Y-linked SNP, tested in 66 known-sex Macquarie perch and two of each sex of three confamilial species, plus amplicon sequencing of 400 bp encompassing the 146-bp region, revealed that the few sex-linked positions differ between species and between Macquarie perch populations. This indicates sex-chromosome lability in Percichthyidae, supported by nonhomologous scaffolds containing sex-linked loci for Macquarie- and golden perches. The present resources facilitate genomic research in Percichthyidae, including formulation of hypotheses about candidate genes of interest such as transcription factor SOX1b that occurs in the 275-kb scaffold ~38 kb downstream of the 146-bp region containing seven XY-gametologous loci. Sex-linked markers will be useful for determining genetic sex in some populations and studying sex chromosome turnover.