Members of the diatom family rhopalodiaceae possess cyanobacteria-derived intracellular structures called spheroid bodies (SBs) that very likely carry out nitrogen fixation. Due to the shortage of molecular data from SBs and rhopalodiacean diatoms, it remains unclear how SBs were established and spread in rhopalodiacean diatoms. We here amplified the small subunit ribosomal DNA sequences from both host and SB in three rhopalodiacean diatom species, Epithemia turgida, E. sorex, and Rhopalodia gibba. Phylogenetic analyses considering these new sequences clearly indicate that the SBs were acquired by a common ancestor of rhopalodiacean diatoms and have been retained during host speciation.