The condition of mtDNA in hair shafts preserved in a museum was examined using 30 study skins of masked palm civets, Paguma larvata (Viverridae), collected between 1924 and 2011. Comparisons of extracts from fresh and burnt alum-fixed hair shafts showed that burnt alum, which is commonly used in taxidermy, had no harmful effect on the amount of total DNA and lengths of the mtDNA fragments. Burnt alum-fixed hair shafts had a tendency to develop a small degree of melanin hindering PCR amplification compared with fresh hair shafts, although that observation was not supported statistically (Wilcoxon signed-rank test, Z = -0.183, P = 0.855). However, the amount of total DNA decreased after preparation of specimen in an exponential relation (regression of log DNA amount with year, regression analysis, F = 7.065, P = 0.013). Nevertheless, the oldest specimen, collected in 1924, yielded 1341.5 ng of DNA per 100 hair shafts, which was sufficient for PCR amplification. In addition, the mtDNA fragment length and amount of melanin in the hair shaft were not significantly correlated with the passage of time after preparation (F = 0.244, P = 0.625 for mtDNA fragment length; F = 0.039, P = 0.845 for the amount of melanin). Therefore, hair shafts prepared and preserved by chemical treatment in museums are good sources of mtDNA and useful for genetic analysis.