Southern blotting is a common method used for the study of gene organization. Current methods of DNA transfer for Southern blotting, however, can be inefficient for high concentration agarose gels. Here, we report a method for high-performance Southern blotting of short DNA fragments such as nucleosomal DNAs by using a discontinuous agarose zone gel. The results show that sharp and well-resolved fractionation of short DNA fragments comparable to that from a high-concentration agarose gel could be obtained using a low-concentration agarose gel with a small zone of high-concentration agarose, and that the resulting DNA transfer is highly efficient and rapid.