This study describes an optimized DNA extraction protocol targeting ultrashort DNA molecules from single rootless hairs. It was applied to the oldest samples available to us: locks of hairs that were found in relics associated with the Romanov family. Published mitochondrial DNA genome sequences of Tsar Nicholas II and his wife, Tsarina Alexandra, made these samples ideal to assess this DNA extraction protocol and evaluate the types of genetic information that can be recovered by sequencing ultrashort fragments. Using this method, the mtGenome of the Tsarina’s lineage was identified in hairs that were concealed in a pendant made by Karl Fabergé for Alexandra Feodorovna Romanov. In addition, to determine if the lock originated from more than one individual, two hairs from the locket were extracted independently and converted into Illumina libraries for shotgun sequencing on a NextSeq 500 platform. From these data, autosomal SNPs were analyzed to assess relatedness. The results indicated that the two hairs came from a single individual. Genetic testing of hairs that were found in the second artifact, a framed photograph of Louise of Hesse-Kassel, Queen of Denmark and maternal grandmother of Tsar Nicholas II, revealed that the hair belonged to a woman who shared Tsar Nicholas’ maternal lineage, including the well-known point heteroplasmy at position 16169.