The present study demonstrates a reinvestigation of the mitochondrial DNA sequence heteroplasmy, which was previously found by the use of nested polymerase chain reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach was used for the amplification of mitochondrial DNA and a phylogenetic analysis was applied to both data sets for the verification of the authenticity of sequences. The comparative analysis of the sequencing results obtained from the same hair DNA extracts - but using two different techniques - shows that direct mitochondrial DNA amplification results in a considerably lower number of mixed positions. The majority of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic mutations observed in four extracts after nested PCR significantly differs from the pattern of natural mutations. Some of these rare polymorphisms should be revised as inconsistent with phylogenetic expectations. The results of the present study contribute to the earlier reports by indicating that phylogenetic analysis is an effective tool in a posteriori quality check of mitochondrial DNA data.