Understanding sex-specific biology can aid conservation management. But understanding genomic sex differences of monomorphic fish species and developing molecular sexing assays is challenged by their diverse sex-determination systems. To facilitate research on Percichthyid fish, predominant in the Australian freshwater biota, we report whole genome sequences and annotations of the endangered Macquarie perch Macquaria australasica and its sister species, the golden perch M. ambigua. To identify sex-linked loci, we conducted whole genome resequencing on 100 known-sex Macquarie perch. In-silico pool-seq comparisons revealed few sex differences, but a 275-Kb SOX-containing scaffold was enriched for gametologous loci- homozygous in females, heterozygous in males. Within this scaffold we reconstructed X- and Y-linked 146-bp haplotypes containing 5 sex-linked SNPs, ~38 Kb upstream of SOX, and developed a PCR-RFLP sexing assay targeting the Y-linked allele of one SNP. We tested this assay in a panel of known-sex Macquarie perch, and smaller panels of three other confamilial species. Amplicon sequencing of 400 bp encompassing the 146-bp region revealed that the few sex-linked positions differ interspecifically, and within Macquarie perch such that its sexing test approached 100% reliability only for the populations used in assay development. Similarly, Macquarie- and golden perch genome-wide DArTseq SNPs revealed different sex-linked loci across non-homologous scaffolds. Overall, we identified 22 sex-linked SNPs in Macquarie perch in a predominantly XX/XY system in which females are homozygous at all 22, and males are heterozygous at 2 or more. The resources here will facilitate multi-locus sexing assays for both species and research on Percichthyid biology.