We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations.