Abstract In most eukaryotic genomes, tandemly repeated copies of 5S rRNA genes are clustered outside the nucleolus organizer region (NOR), which normally encodes three other major rRNAs: 18S, 5.8S, and 28S. Our analysis of turtle rDNA sequences has revealed a 5S rDNA insertion into the NOR intergenic spacer in antisense orientation. The insertion (hereafter called NOR-5S rRNA gene) has a length of 119 bp and coexists with the canonical 5S rDNA clusters outside the NOR. Despite the ∼20% nucleotide difference between the two 5S gene sequences, their internal control regions for RNA polymerase III are similar. Using the turtle Trachemys scripta as a model species, we showed the NOR-5S rDNA specific expression in oocytes. This expression is concurrent with the NOR rDNA amplification during oocyte growth. We show that in vitellogenic oocytes, the NOR-5S rRNA prevails over the canonical 5S rRNA in the ribosomes, suggesting a role of modified ribosomes in oocyte-specific translation. The orders Testudines and Crocodilia seem to be the only taxa of vertebrates with such a peculiar rDNA organization. We speculate that the amplification of the 5S rRNA genes as a part of the NOR DNA during oogenesis provides a dosage balance between transcription of all the four ribosomal RNAs while producing a maternal pool of extra ribosomes. We further hypothesize that the NOR-5S rDNA insertion appeared in the Archelosauria clade during the Permian period and was lost later in the ancestors of Aves.