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The Aesculapian snake (Zamenis longissimus) is distributed in Central and Southern Europe, the Balkans, Anatolia, and Iran, but had a wider mid-Holocene distribution into Northern Europe. To investigate the genetic affinity of a Danish population that went extinct in historical times, we analysed three ethanol-preserved individuals dating back to 1810 using a silica-in-solution ancient DNA extraction method, combined with next-generation sequencing. Bioinformatic mapping of the reads against the published genome of a related colubrid snake revealed that two of the three specimens contained endogenous snake DNA (up to 8.6% of the reads), and this was evident for tooth, bone, and soft tissue samples. The DNA was highly degraded, observed by very short average sequence lengths (<50 bp) and 11-15% C to T deamination damage at the first 50 position. This is an effect of specimen age, combined with suboptimal, and possibly damaging, molecular preservation conditions. Phylogeographic analyses of a 1638 bp mtDNA sequence securely placed the two Danish Aesculapian snakes in the Eastern (Balkan glacial refugium) clade within this species, and revealed one previously-undescribed haplotype. These results provide new information on the past distribution and postglacial re-colonization patterns of this species. [ABSTRACT FROM AUTHOR]