Population dynamics can be analysed at the spatial and temporal scales by using museum specimens. DNA-based sex determination systems can be applied to many biological samples, including preserved museum specimens. Systems applicable to historical DNA of non-mustelid species were adapted to obtain short but sufficiently informative fragments of mustelid sex-linked genes. Here, I used the two most popular methods — SRY amplification and polymorphism analysis of ZFX/ZFY genes — to determine sex of individuals belonging to several mustelid species. Positive results of SRY amplification (115 bp) in males and the lack of the product in females were confirmed by the analysis of sex-specific mutations in the ~90 bp fragment of ZFX/Y for five species: Neovison vison, Mustela putorius, Mustela erminea, Lutra lutra, and Meles meles. For Martes foina, the analysed ZFX/Y fragment did not differ between male and female samples. For Mustela nivalis, only a male sample was available and no reference ZF sequences were found.