A new approach is presented for preparative, continuous flow fractionation of sub-10-kbp DNA fragments, which exploits the variation in the field-dependent mobility of the DNA molecules based on their length. Orthogonally pulsed electric fields of significantly different magnitudes are applied to a microchip filled with a sieving matrix of 1.2% agarose gel. Using this method, we demonstrate a high-resolution separation of 0.5, 1, 2, 5, and 10 kbp DNA fragments within 2 min. During the separation, DNA fragments are also purified from other ionic species. Preparative fractionation of sub-10-kbp DNA molecules plays an important role in second-generation sequencing. The presented device performs rapid high-resolution fractionation and it can be reliably manufactured with simple microfabrication procedures.