Mound building was a common practice in the Midwestern United States among the Adena and Middle Mississippian tribes from approximately 500 B.C. to 1500 A.D. Though they varied greatly in size, shape, and complexity between cultures, the mounds served a common purpose as means to bury the deceased. Mounds representing both cultures have been found dispersed throughout Indiana, in areas such as Randolph County, Henry County, Madison County, Knox County, and Vanderburgh County.Of particular interest in this investigation is an Adena burial plot, known as Windsor Mound, which is 2000 yrs old and located in neighboring Randolph County. An amateur excavation of the mound from 1986 to 1988 unearthed the fragmented remains of 44 individuals and several artifacts, all of which were loaned to the Ball State University Anthropology Department. Documentation of the excavation was poor and the exact location of artifacts and remains within the mound were not recorded, leaving anthropologists with many unanswered questions that could only be answered through genetic analysis. Pressing questions were the degree to which the 44 individuals were related and which of the five Native American lineages (haplotypes) they belonged to.For this analysis, dentin was recovered from the teeth of three Native American individuals exhumed from Windsor Mound. Two of the individuals (87.17.5 and 87.17.10) were found in the lower mound portion (70 B.C.), while the third individual (87.17.22) was found in the upper mound cap (1180 A.D.). Ancient DNA (aDNA) was extracted from each dentin sample, yielding an average of 0.072 ug/mg of dentin. A highly polymorphic portion of the mitochondria) DNA control region (nt 16,049 - nt 16,221) termed hypervariable region 1 (HVI) was amplified via PCR to generate 172 bp amplicons which were cloned into a plasmid vector. Following a transformation, 10 clones from each individual were sequenced and aligned to identify consistent mutations, as opposed to random post-mortem damage that may have occurred.Sequencing of the HVI region for Individual 87.17.5 revealed a T —~ C base substitution at nt 16,189, a C — T substitution at nt 16,192, and a deletion at nt 16,203. Identical mutations were seen in Individual 87.17.22 from the upper mound cap. The alignment for Individual 87.17.10 revealed a unique T --~ C mutation at nt 16,126 as well as the deletion at nt 16,203. A literature search revealed that the substitution at nt 16,189 is specific to Native American 1-laplogroups B and X. Haplogroup B first appeared in central Asia 60,000 yrs ago while Haplogroup X appeared in western Asia 30,000 yrs ago. The substitution at nt 16,192 is indicative of East Asian origin and specific to Japanese populations. To our knowledge the deletion at nt 16,203 has not been reported before in a Native American, thus its presence was thought to indicate kinship between the individuals. However, an alignment of the ancient consensus sequences to that of the three investigators revealed that the deletion was present in two of the modern samples and was not novel. Nothing is known about the substitution at nt 16,126. Further sequencing downstream of nt 16,221 is needed to identify additional mutations characteristic of Haplogroup B or X and a novel kinship marker. / Department of Biology