Extraction of DNA from Mycoplasma cultured on agar medium is difficult because the plasticity of these microorganisms enables agar penetration. This eventually causes cell loss during harvesting of colonies from the agar surface. Here, we used the GenElute™ gel extraction kit, which is usually used to purify polymerase chain reaction products, for extracting DNA from Mycoplasma. We compared the DNA extraction efficiency of the GenElute™ gel extraction kit from Mycoplasma cynos cultured in agar medium with four other DNA extraction methods. The results were evaluated based on the purity and amount of DNA obtained from one Mycoplasma colony. Eight strains of Mycoplasma cynos isolated from the broncho-alveolar lavage fluid of dogs were used. The GenElute™ gel extraction protocol was the most efficient among all the methods tested in this study as it yielded the highest amount and the purest quality of DNA (199.3 ± 0.744 ng/μl) from a single colony. Among the methods tested, the GenElute™ gel extraction method is the most rapid, sensitive, and simple method for DNA extraction from Mycoplasma. This procedure may also prove useful for extracting DNA from other Mycoplasma species.