Hair is a prominent physiological feature of many animals that can be easily and non-invasively sampled. However, most previous studies have focused on human hair, and animal hair has not been widely used as a DNA source in genetic and evolutionary studies. Analysing the DNA quality of animal hair is more challenging than that of human hair because it varies across different species and is influenced by different storage conditions. To address this gap in our knowledge, this study systematically evaluated the DNA quality of various animal hair samples. Our findings show that lightly medullated hairs, which are typical in carnivores, yield greater amounts of DNA and have better sequencing results than highly medullated hairs, which are typical in ungulate herbivores. Furthermore, the roots and middle hair were more productive than the upper hair parts. Tanning and storage time impair DNA yield and sequencing quality and increase nucleotide damage. Moreover, for certain historical specimens and field samples, genomic DNA was more successfully obtained from hair samples than from skin samples. This study provides data and theoretical support for improving the use of animal hair in genetic research.