In the vast majority of Animalia genomes, the 5S rRNA gene repeats are located on chromosomes outside of the 45S rDNA arrays of the nucleolar organiser (NOR). We analysed the genomic databases available and found that a 5S rDNA sequence is inserted into the intergenic spacer (IGS) between the 45S rDNA repeats in ten species of the family Nototheniidae (Perciformes, Actinopterigii). We call this sequence the NOR-5S rRNA gene. Along with Testudines and Crocodilia, this is the second case of a close association between four rRNA genes within one repetitive unit in deuterostomes. In both cases, NOR-5S is oriented opposite the 45S rDNA. None of the three nucleotide substitutions compared to the canonical 5S rRNA gene influenced the 5S rRNA secondary structure. In transcriptomes of the Patagonian toothfish, we only found NOR-5S rRNA reads in ovaries and early embryos, but not in testis or somatic tissues of adults. Thus, we consider the NOR-5S gene to be a maternal-type 5S rRNA template. The colocalization of the 5S and 45S ribosomal genes appears to be essential for the equimolar production of all four rRNAs in the species that show rDNA amplification during oogenesis. Most likely, the integration of 5S and NOR rRNA genes occurred prior to Nototheniidae lineage diversification.