Two recent studies have presented conflicting views on variation present within the 294 base third domain of the 12S rRNA gene in the genus Drosophila, and in D. pseudoobscura in particular. One study suggested that this gene is highly invariant across the genus, while another recovered 22 distinct haplotypes from 22 strains of D. pseudoobscura. We have sequenced this gene in numerous lines of D. pseudoobscura and its relatives, noting only two haplotypes in the third domain, and we failed to confirm any of the published sequences. Second, we note that the published sequence divergence between strains of D. pseudoobscura was as great as that documented between distantly related Drosophila species. Third, we show that the published polymorphisms of this region within D. pseudoobscura would disrupt the secondary structure of the resulting molecule. We conclude that the published 12S rRNA sequences of D. pseudoobscura do not accurately reflect variability of the functional gene, and that this gene is relatively invariant in D. pseudoobscura and D. persimilis.