Hair roots constitute a preferred sample type for genetic studies in horses because their collection is less invasive than blood sampling. Hair is also easier to store for long periods of time. The amount of starting material and DNA extraction method are crucial factors. In our study, different DNA extraction methods were evaluated using mane hair samples obtained from Czech sport horses (CS). The adapted protocol employing the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) was found to be the most effective.