A fast, simple, and reliable mini-prep method for the extraction of DNA from Gossypium species and cultivars has been developed. This small-scale method is cetyltrimethylammonium bromide (cTAB)-based, and it extracts DNA from one to three folded or nearly unfolded young leaves processed in a 1.5 mL tube with 0.5 mL of extraction buffer and homogenized by an electric drill. Compared with the macro-prep cTAB method, the improved mini-prep method is highly efficient and much cheaper in terms of time, chemical use, and labor input. Easily 200 samples per day can be processed by a single person. The DNA yield is greater (60 μg per 50-100 mg of fresh leaf tissue) than that obtained from the macro-prep method (50 μg from 5 g of fresh leaf tissue) and it provides DNA for 3000 to 6000 polymerase chain reactions (PCRs). The DNA quality is sufficient for PCR-based and endonuclease restriction marker analysis.