Analyzing microsatellite instability (MI) in malignant tumors is thought to be useful for screening cancer patients to identify those patients with a higher risk of developing second malignant tumors. In this paper, we report a new, accurate, and efficient method of detecting MI using an automated fluorescent DNA sequencer and a computer that automatically calculates the size, height, and area of each fluorescent product, making it possible to assess MI more accurately and more rapidly. The primers for amplification of each microsatellite locus are labeled by two different fluorescent dyes, rox (red) and fam (blue). The rox-labeled primer was used for the tumor, whereas the fam-labeled primer was used for the corresponding normal tissue. Two amplified products from both the tumor and the normal tissue were co-loaded into a single lane of the sequencing gel and were analyzed. MI could be detected based on the presence of different waving patterns. Furthermore, several loci could also be analyzed simultaneously for MI in a single lane. Using this method, we examined the frequency of MI in gastric cancer. The results showed that 5 of 22 (22.7 %) gastric cancers were MI-positive, which corresponds to the findings of previous reports that used the radioisotopic method. The improved method may open up the possibility of performing routine examination of MI in many cancer patients and offers hope for the potential clinical application of Ml analysis as a follow-up evaluation of cancer patients.