Objectives The aim of this study was to explore the mitochondrial variability in the Yanomami population to reconstruct its demographic history and explore its genetic composition in relation to its cultural and linguistic features. Methods A total of 174 human head hair shafts ‐collected in 1958‐ belonging to individuals from a Yanomami group living in Santa Isabel, Brazil, were analyzed. Automated extraction of the hairs was performed, and several methods were applied to optimize the analysis of the degraded DNA. The mtDNA hypervariable segments I–II, along with the 9‐bp COII‐tRNALys deletion, were investigated. Using published data from the Yanomami and other Amazonian populations, several statistical analyses were carried out to explore the genetic variability within the study population. Results Ninety eight percent of the mitochondrial DNA (mtDNA) sequences analyzed belonged to Native American haplogroups, while 2% belonged to African haplogroups. Compared with the Yanomami groups previously studied, the Santa Isabel sample seemed more genetically similar to other Amazonian populations. Conclusions Among the Yanomami samples studied to date, the Santa Isabel Yanomami show a higher genetic heterogeneity. This could be due to gene flow with non‐Yanomami populations, as well as to the introduction of new mitochondrial haplotypes by gold miners. In both cases, the geographic location of Santa Isabel might have made this Yanomami village less isolated than the others, suggesting that the Rio Negro played a central role in increasing its genetic variability. On the whole, the Yanomami were quite genetically diversified, probably mirroring their great linguistic heterogeneity. Am. J. Hum. Biol. 28:846–856, 2016. © 2016Wiley Periodicals, Inc.