Chewed and expectorated quids, indigestible stringy fibers from the roasted inner pulp of agave or yucca root, have proven resilient over long periods of time in dry cave environments and correspondingly, although little studied, are common in archaeological archives. In the late 1960s, thousands of quids were recovered from Mule Spring Rockshelter (Nevada, USA) deposits and stored without consideration to DNA preservation in a museum collection, remaining unstudied for over fifty years. To assess the utility of these materials as repositories for genetic information about past inhabitants of the region and their movements, twenty-one quids were selected from arbitrary excavation depths for detailed analysis. Human mitochondrial DNA sequences from the quids were amplified by PCR and screened for diagnostic single nucleotide polymorphisms. Most detected single nucleotide polymorphisms were consistent with recognized Native American haplogroup subclades B2a5, B2i1, C1, C1c, C1c2, and D1; with the majority of the sample set consistent with subclades C1, C1c, and C1c2. In parallel with the DNA analysis, each quid was radiocarbon dated, revealing a time-resolved pattern of occupancy from 347 to 977 calibrated years before present. In particular, this dataset reveals strong evidence for the presence of haplogroup C1/C1c at the Southwestern edge of the US Great Basin from ~670 to 980 cal YBP, which may temporally correspond with the beginnings of the so-called Numic Spread into the region. The research described here demonstrates an approach which combines targeted DNA analysis with radiocarbon age dating; thus enabling the genetic analysis of archaeological materials of uncertain stratigraphic context. Here we present a survey of the maternal genetic profiles from people who used the Mule Spring Rockshelter and the historic timing of their utilization of a key natural resource.