Two genes, that is, 5S ribosomal sequences and antifreeze glycoprotein (AFGP) genes, were mapped onto chromosomes of eight Antarctic notothenioid fish possessing a X1X1X2X2/X1X2Y sex chromosome system, namely,Chionodraco hamatusandPagetopsis macropterus(family Channichthyidae),Trematomus hansoni,T. newnesi,T. nicolai,T. lepidorhinus, andPagothenia borchgrevinki(family Nototheniidae), andArtedidraco skottsbergi(family Artedidraconidae). Through fluorescencein situhybridization (FISH), we uncovered distinct differences in the gene content of the Y chromosomes in the eight species, withC. hamatusandP. macropterusstanding out among others in bearing 5S rDNA and AFGP sequences on their Y chromosomes, respectively. Both genes were absent from the Y chromosomes of any analyzed species. The distinct patterns of Y and non-Y chromosome association of the 5S rDNA and AFGP genes in species representing different Antarctic fish families support an independent origin of the sex heterochromosomes in notothenioids with interesting implications for the evolutionary/adaptational history of these fishes living in a cold-stable environment.