A mitochondrial DNA (mtDNA) haplogroup association study carried out in 101 southern Portuguese males with oligozoospermia showed to be negative when comparing with a geographically matching control sample. Misleading positive association signs were however obtained when using other control samples from the same country. This shows that mtDNA population substructure can also introduce spurious signs in haplogroup association studies, as previously reported for Y-chromosome. However, our data do not exclude the probability that a particular mtDNA mutation contributes significantly to the reduction of sperm production, but indeed precludes the hypothesis of a significant association between such a mutation and specific haplogroup.