A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-mu m plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of Phi X174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 mu m (corresponding to plate numbers from 4.86 x 10(6) to 1.36 x 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n = 8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.