When mitochondrial DNA (mtDNA) heteroplasmies are detected, they often confound forensic identification, especially if they are the result of poor biological sampling. In this study, we determined the ratio of heteroplasmy in samples that were amplified from a very small amount of template mtDNA or a few cells using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis. As a result, more than half of the detected sequences (i.e., 17/20, 15/20, and 14/20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. Additionally, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These results indicate the risks associated with using highly sensitive mtDNA techniques in forensic investigations because of the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample.