The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.