We describe a quick and systematic optimization of molecular weights (MWs) and concentrations of a series of mixed poly(ethylene oxide) (PEO) matrices for separating specific double-stranded DNA fragments on polymethylmethacrylate-based microchips by using an orthogonal design (ORD) approach. The mixed matrices are composed of PEOs in four MW ranges (M-w 8 x 10(6), 1 x 10(6), 4 x 10(5), and 1 x 10(5)) with varying concentration ratios. In the mixed solutions, PEO with an intermediate MW of 4 x 105 is found to be a dominant factor for separating small DNA fragment pairs (e.g., 82 and 88 bp), while PEO with a high MW of 8 x 106 plays an important role in separating intermediate and large fragments (e.g., 271 and 281 bp, 506 and 517 bp, 7 and 10 kbp). High-concentration PEO mixtures give better resolution for short fragments, while dilute PEO mixtures show better resolution for long fragments. The optimized matrices are suitable for high-resolution separation of multiplex polymerase chain reaction-amplified products and restriction digest fragments ranging in size from 20 bp to 40 kbp within 4 min at a constant field strength of 177 V/cm. The experimental results indicate the robustness and speediness of the ORD to screen the contribution of PEO MWs and to tune optimally the PEO concentration ratio of different MWs with reference to the performance of specific DNA fragments separated.