We compared two different PCR strategies for the amplification of mtDNA hypervariable region 1 (HV1) with regard to the detection and interpretation of point mutation heteroplasmy in human hair roots. We monitored the level of detected heteroplasmy using direct sequence analysis. PCR amplifications were performed in duplicate on each hair root, using 62 cycles of nested PCR versus 35 cycles of direct PCR. As a previous publication reported different sensitivities of heteroplasmy detection based on the number of PCR cycles used, we were interested in whether and how different PCR amplification strategies would impact sequence quality and the detection of point heteroplasmy. We identified 12 out of 93 hair roots as heteroplasmic (7 out of 31 persons) with direct PCR, whereas 2 of these heteroplasmic events could not be identified with the nested PCR approach. Generally, the quality of the sequence electropherograms in terms of background noise was significantly lower for the nested PCR amplification strategy, leading to ambiguous results in some of the nucleotide positions. Thus, the ability to clearly distinguish a genuine mixture of two nucleotides from background noise at a heteroplasmic position was substantially greater with direct PCR amplification, which generally resulted in higher quality sequence electopherograms.