Metaphase chromosomes of the scallop Hinnites distortus were analyzed using Giemsa staining, chromosome measurements, silver staining, one- and two-color fluorescent in situ hybridization (FISH) ribosomal DNA (rDNA) probes, and 4',6-diamidino-2-phenylindole (DAPI) banding compatible with in situ hybridization. The karyotype (2n = 38) consists of three submetacentric-metacentric, one submetacentric, one subtelocentric-submetacentric, and 14 subtelocentric pairs. The 18S-28S rDNA maps at the centromeric level of two subtelocentric pairs, but not more than two nucleolus organizer region (NOR)-bearing chromosomes were transcriptionally active. The 5S rDNA seems to show a conventional tandem arrangement with a repeat unit of about 450 bp and it maps at the pericentromeric region of the long arm of one subtelocentric pair. Two-color FISH demonstrated that 18S-28S rDNA and 5S rDNA are not syntenic. Sequential FISH/Giemsa staining and subsequent chromosome pairing allow us to propose that pairs 9 and 12 carry the 18S-28S rDNA and pair 13 carries the 5S rDNA. All chromosomes are characterized as containing constitutive heterochromatin at the centromeric region. The data provided are the first contribution toward construction of the molecular karyotype of H. distortus and will be useful in assessing evolutionary relationships within scallops.