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Collectively called zooxanthellae, photosynthetic dinoflagellates in the family Symbiodiniaceae are typical endosymbionts that unequivocally mediate coral responses to environmental changes. Symbiodiniaceae are genetically diverse, encompassing at least nine phylogenetically distinct genera (clades A–I). The ribosomal internal transcribed spacer 2 (ITS2) region is commonly utilized for determining Symbiodiniaceae diversity within clades. However, ITS2 is often inadvertently interpreted together with the tailing part of the ribosomal RNA genes (5.8S and 28S or equivalent), leading to unresolved taxonomy and equivocal annotations. To overcome this hurdle, we mined in GenBank and expert reference databases for ITS2 sequences of Symbiodiniaceae having explicit boundaries with adjacent rRNAs. We profiled a Hidden Markov Model of the ITS2‐proximal 5.8S‐28S rRNA interaction, which was shown to facilitate the delimitation of Symbiodiniaceae ITS2 from GenBank, while considerably reducing sequence ambiguity and redundancy in reference databases. The delineation of ITS2 sequences unveiled intra‐clade sequence diversity and inter‐clade secondary structure conservation. We compiled the clean data into a non‐redundant database that archives the largest number of Symbiodiniaceae ITS2 sequences known to date with definite genotype/subclade representations and well‐defined secondary structures. This database provides a fundamental reference catalog for consistent and precise genotyping of Symbiodiniaceae and a tool for automated annotation of user‐supplied sequences.

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