Recovered and amplified ancient DNA (aDNA), from a historically documented 19th century Upper Canadian pioneer cemetery produced genotypes that were used to infer a past societal kinship system. While the results from multiplex short tandem repeat (STR) amplifications showed an unreliable polymerase chain reaction (PCR) product, a single locus HUMTH01 analysis yielded reproducible data and an allelic frequency pattern not statistically different from modern populations. Mitochondrial (mt) DNA HVR II data showed that a combined cemetery database exhibited reduced haplotype diversity indicators, as well as clusters of probable maternally related burials. The chronological persistence and replacement of mtDNA clusters approximately every two generations suggests a patrilineal/patrilocal kinship structure from a virilocal burial program for the Harmony Road cemetery. Through the integration of the aDNA analysis with archaeological material culture, historic records, and other ethnohistoric sources of information, this conclusion is supported. In this study persisting patrilineally inherited surnames act as a surrogate for aDNA Y-chromosome haplotype analysis. These results suggest that aDNA applications on aggregate skeletal collections where sparse, or no ethnological or historical documentation exists, may result in incorrect population history inferences if the presence of a kinship interment bias is not considered.