AbstractMitochondrial cox1 689 bp barcodes are routinely used for identification of Tetrahymena species. Here, we examine whether two shorter nuclear sequences, the 5.8S rRNA gene region and the intergenic region between H3 and H4 histone genes, might also be useful either singly or in combination with each other or cox1. We obtained sequences from ~300 wild isolates deposited at the Tetrahymena Stock Center and analyzed additional sequences obtained from GenBank. The 5.8S rRNA gene and portions of its transcribed flanks identify isolates as to their major clade and uniquely identify some, but not all, species. The ~330 bp H3/H4 intergenic region possesses low intraspecific variability and is unique for most species. However, it fails to distinguish between two pairs of common species and their rarer counterparts, and its use is complicated by the presence of duplicate genes in some species. The results show that while the cox1 sequence is the best single marker for Tetrahymena species identification, 5.8S rRNA, and the H3/H4 intergenic regions sequences are useful, singly or in combination, to confirm cox1 species assignments or as part of a preliminary survey of newly collected Tetrahymena. From our newly collected isolates, the results extend the biogeographical range of T. shanghaiensis and T. malaccensis and identify a new species, Tetrahymena arleneae n. sp. herein described.