Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on. Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in < 90 min.