Efficient capillary electrophoretic STR analysis requires the rapid, reproducible, and robust separation of DNA fragments with reasonable capillary longevity. This is currently done by using proprietary commercial polymeric sieving matrixes specifically developed for this separation. Although effective, these matrixes are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths. This requires additional time to achieve the separation and minimize the potential extrapolation to other miniaturized platforms. For these reasons, other commercially available polymeric separation matrixes are being systematically evaluated for use in microdevices. The current report describes the evaluation of polyethylene oxide (PEO) for the separation of STR fragments in capillaries, with a view toward use in the microchip platform and as a possible alternative to commercially available sieving matrixes. Accurate, reproducible separation of single base pair differences is reported using a low-viscosity, denaturing solution of low-molecular-weight PEO in short capillaries. In addition, the effects of capillary temperature, buffer composition, polymer weight and concentration, and capillary length are described. Resolution was found to be better than results obtained with the use of conventional commercial polymers, with significantly shorter run times. The feasibility and demonstration of inclusion of this separation matrix in a typical microdevice are also discussed. 6 figures and 35 references