The species-rich diatom family Chaetocerotaceae is common in the coastal marine phytoplankton worldwide where it is responsible for a substantial part of the primary production. Despite its relevance for the global cycling of carbon and silica, many species are still described only morphologically, and numerous specimens do not fit any described taxa. Nowadays, studies to assess plankton biodiversity deploy high throughput sequencing metabarcoding of the 18S rDNA V4 region, but to translate the gathered metabarcodes into biologically meaningful taxa, there is a need for reference barcodes. However, 18S reference barcodes for this important family are still relatively scarce. We provide 18S rDNA and partial 28S rDNA reference sequences of 443 morphologically characterized chaetocerotacean strains. We gathered 164 of the 216 18S sequences and 244 of the 413 28S sequences of strains from the Gulf of Naples, Atlantic France, and Chile. Inferred phylogenies showed 84 terminal taxa in seven principal clades. Two of these clades included terminal taxa whose rDNA sequences contained spliceosomal and Group IC1 introns. Regarding the commonly used metabarcode markers in planktonic diversity studies, all terminal taxa can be discriminated with the 18S V4 hypervariable region; its primers fit their targets in all but two species, and the V4-tree topology is similar to that of the 18S. Hence V4-metabarcodes of unknown Chaetocerotaceae are assignable to the family. Regarding the V9 hypervariable region, most terminal taxa can be discriminated, but several contain introns in their primer targets. Moreover, poor phylogenetic resolution of the V9 region affects placement of metabarcodes of putative but unknown chaetocerotacean taxa, and hence, uncertainty in taxonomic assignment, even of higher taxa.