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The production of lumpfish (<i>Cyclopterus lumpus</i>) has become crucial in controlling sea lice levels in salmonid aquaculture. To improve their breeding, there is a need for early sex identification. The genomic region containing the <i>anti-Müllerian hormone</i> (<i>amh</i>) gene was suggested as the candidate master sex-determining gene in lumpfish. However, the genome of lumpfish contains three copies of <i>amh</i> with ambiguous sex specificity, designated <i>amh1</i>, <i>amh2</i>, and <i>amh3</i>. The study aims to analyse the male-specific region between these <i>amh</i> paralogues for its application as a sex marker. In this study, we utilised polymerase chain reaction (PCR)-based assays to identify the male-specific <i>amh</i> markers in lumpfish and estimate the length of the male-specific region in the lumpfish genome. Our results indicate that a specific genomic region of approximately 27 kilobases (kb), encompassing <i>amh1</i> and <i>amh2</i> genes, exhibits male specificity, whereas <i>amh3</i> is present in both sexes. The developed PCR-based genetic sex identification assays targeting <i>amh1</i> and <i>amh2</i> exhibited over 97% concordance with phenotypic records. Further experiments in other members of the Cyclopteridae: <i>Aptocyclus ventricosus</i>, <i>Eumicrotremus taranetzi</i>, and <i>E. asperrimus</i> revealed male-specific <i>amh</i> genome region only in <i>A. ventricosus</i>. Phylogenetic analyses using the available Cyclopteridae <i>amh</i> sequences suggest that male-specific <i>amh</i> arose early in the Cyclopteridae lineage. Our findings, along with the development of the PCR test, hold great promise for the field of lumpfish aquaculture and will also contribute significantly to future investigations aiming to enhance our understanding of the sex-determination system and the evolution of sex chromosomes in teleostean fish.