The source of acellular specimens is not infrequently challenged, especially for toxicology specimens, but such specimens may not be amenable to conventional genetic testing to confirm the source. Our evaluation of genomic and mitochondrial DNA (mtDNA) amplicons using polymerase chain reaction (PCR) from centrifuged, filtered, or whole urine specimens demonstrated higher sensitivity of detection of mtDNA than genomic DNA and a higher detection rate for the mtDNA markers than genomic markers in all sample sets. The mitochondrial amplicons were sequenced to identify specific single nucleotide polymorphisms (SNPs). Subsequently, a real-time PCR technique using fluorescence resonance energy transfer (FRET) probes designed to hybridize to the published mtDNA sequence over known SNP locations present within the mitochondrial control region was developed. Our results demonstrate the feasibility of using a FRET-based assay of mitochondrial genotypes with acellular laboratory specimens to screen for specimen mix-ups or to confirm sources of controversial toxicology specimens.