We describe the karyotype, location of nucleolus-organizing regions (NORs) and heterochromatin distribution and composition in the Antarctic chiton Nuttallochiton mirandus. Specimens had a karyotype of 2n = 32 chromosomes, of which two were microchromosomes. Among macrochromosomes, the elements of the first and fourth pair were bi-armed, the others were telocentric. At least six NOR sites were detected with NOR-FISH, but only four were Ag-NOR-banding-positive. The two microchromosomes were essentially euchromatic, while all macrochromosomes exhibited clear pericentromeric C bands that were found to be AT-rich (being quinacrine- and DAPI-positive) and resistant to digestion with AluI and HaeIII. N. mirandus has the largest number of chromosomes (2n = 32) and telocentric elements (26) of all the chiton species studied to date. The karyological results of our study agree with previous molecular data indicating N. mirandus as a sister taxon of Acanthochitona crinita. The karyotypes of the two species could be related as a result of Robertsonian rearrangements. According to the more parsimonious hypothesis, the former would be the primitive karyotype, although other evolutionary events cannot be ruled out.