Mitochondrial DNA (mtDNA) analysis is a fundamental tool in forensic genetics, particularly when biological samples exhibit severe degradation or low nuclear DNA content. Its unique biological characteristics, such as a high copy number per cell, strict matrilineal inheritance, and lack of recombination, enable human identification and reconstruction of maternal lineages in complex contexts, including disaster victim identification, historical cases, and missing persons investigations. This narrative review examines contemporary methodological approaches for investigating the human mitogenome. We discuss recent advancements in extraction and enrichment techniques, emphasizing their efficacy in reducing the interference of nuclear mitochondrial DNA sequences (NUMTs) and enhancing the recovery of informative fragments. Moreover, the shift from traditional Sanger sequencing to Massive Parallel Sequencing (MPS) is examined, as MPS has markedly enhanced the sensitivity and capability of contemporary methods to detect low-frequency heteroplasmies. Additionally, the advent of Third-Generation Sequencing (TGS), exemplified by nanopore platforms, is evaluated, which facilitates the reading of full-length native molecules without the biases introduced by PCR amplification. Despite the interpretive challenges posed by heteroplasmy, contamination, and limitations in population databases, ongoing methodological advances in mitochondrial DNA analysis continue to strengthen its reliability and expand its potential in forensic genetics.

Objectives: Determine the geographic place of origin and maternal lineage of prehistoric human skeletal remains discovered in Puyil Cave, Tabasco State, Mexico, located in a region currently populated by Olmec, Zoque and Maya populations.Materials and Methods: All specimens were radiocarbon (¹⁴C) dated (beta analytic), had dental modifications classified, and had an analysis of 13 homologous reference points conducted to evaluate artificial cranial deformation (ACD). Following DNA purification, hypervariable region I (HVR‐1) of the mitogenome was amplified and Sanger sequenced. Finally, Next Generation Sequencing (NGS) was performed for total DNA. Mitochondrial DNA (mtDNA) variants and haplogroups were determined using BioEdit 7.2 and IGV software and confirmed with MITOMASTER and WebHome softwares.Results: Radiocarbon dating (¹⁴C) demonstrated that the inhabitants of Puyil Cave lived during the Archaic and Classic Periods and displayed tabular oblique and tabular mimetic ACD. These pre‐Hispanic remains exhibited five mtDNA lineages: A, A2, C1, C1c and D4. Network analysis revealed a close genetic affinity between pre‐Hispanic Puyil Cave inhabitants and contemporary Maya subpopulations from Mexico and Guatemala, as well as individuals from Bolivia, Brazil, the Dominican Republic, and China.Conclusions: Our results elucidate the dispersal of pre‐Hispanic Olmec and Maya ancestors and suggest that ACD practices are closely related to Olmec and Maya practices. Additionally, we conclude that ACD has likely been practiced in the region since the Middle‐Archaic Period.