Balamuthia mandrillaris is an opportunistically pathogenic ameba that causes fatal granulomatous amebic encephalitis (GAE) in vertebrates. Previous phylogenetic analyses that included the sequence of a single nuclear small subunit ribosomal RNA gene (18S or ssu rDNA) from this ameba suggested that Balamuthia is closely related to Acanthamoeba, another opportunistically pathogenic amebic genus, which includes multiple ssu rDNA genotypes. We tested whether this also is true for Balamuthia. The nuclear ssu rDNA from 4 isolates and the mitochondrial ssu rDNA from 7 isolates of B. mandrillaris have been sequenced. No variation in the nuclear rDNA sequences and low levels of variation in the mitochondrial rDNA were found. Both gene sequences were consistent with a single genotype for B. mandrillaris. The mitochondrial sequences of B. mandrillaris are unique and should be useful for development of genus-specific diagnostic probes for use with clinical, environmental, and archived specimens.