A combined effect of functional constraints and random mutational events is responsible for the sequence evolution of the human mitochondrial DNA (mtDNA) control region. Most studies targeting this noncoding segment usually focus on its primary sequence information disregarding other informative levels such as secondary or tertiary DNA conformations. In this work, we combined the most recent developments in DNA folding calculations with a phylogenetic comparative approach in order to investigate the formation of intrastrand secondary structures in the human mtDNA control region. Our most striking results are those regarding a new cloverleaf-like secondary structure predicted for a 93-bp stretch of the control region 5′-peripheral domain. Randomized sequences indicated that this structure has a more negative folding energy than the average of random sequences with the same nucleotide composition. In addition, a sliding window scan across the complete mitochondrial genome revealed that it stands out as having one of the highest folding potential. Moreover, we detected several lines of evidence of both negative and positive selection on this structure with high levels of conservation at the structure-relevant stem regions and the occurrence of compensatory base changes in the primate lineage. In the light of previous data, we discuss the possible involvement of this structure in mtDNA replication and/or transcription. We conclude that maintenance of this structure is responsible for the observed heterogeneity in the rate of substitution among sites in part of the human hypervariable region I and that it is a hot spot for the 3′ end of human mtDNA deletions.