IntroductionBalamuthia(B.)mandrillarisis a free-living amoeba that can cause rare yet fatal granulomatous amoebic encephalitis (GAE). However, efficacious treatment for GAE is currently unavailable, especially when genomic studies onB. mandrillarisare limited.MethodsIn this study,B. mandrillarisstrain KM-20 was isolated from the brain tissue of a GAE patient, and its mitochondrial genome wasde novoassembled using high-coverage Nanopore long reads and Illumina short reads.Results and DiscussionPhylogenetic and comparative analyses revealed a range of diversification in the mitochondrial genome of KM-20 and nine otherB. mandrillarisstrains. According to the mitochondrial genome alignment, one of the most variable regions was observed in the ribosomal protein S3 (rps3), which was caused by an array of novel protein tandem repeats. The repeating units in therps3protein tandem region present significant copy number variations (CNVs) amongB. mandrillarisstrains and suggest KM-20 as the most divergent strain for its highly variable sequence and highest copy number inrps3. Moreover, mitochondrial heteroplasmy was observed in strain V039, and two genotypes ofrps3are caused by the CNVs in the tandem repeats. Taken together, the copy number and sequence variations of the protein tandem repeats enablerps3to be a perfect target for clinical genotyping assay forB. mandrillaris. The mitochondrial genome diversity ofB. mandrillarispaves the way to investigate the phylogeny and diversification of pathogenic amoebae.