Microsatellite instability (MSI) plays an important biological role in various types of cancers, and especially in colorectal cancers. This study aimed to develop a simple, efficient, new method for robust MSI analysis. DNA was extracted from 175 (105 proximal colon and 70 distal colorectal) cancer samples and matched normal tissues, and five Bethesda microsatellite markers (BAT-25, BAT-26, D5S346, D2S123, and D17S250) were examined for MSI by denaturing high-performance liquid chromatography (DHPLC) analysis at a temperature of 50 degrees C and a flow rate of 0.9 ml/min. It took just 9 min per PCR product to determine MSI or microsatellite stability (MSS) using the new protocol. The DHPLC results were confirmed with conventional gel-based electrophoresis and capillary-based sequencing method. Of 175 samples, 45 (26%) showed high microsatellite instability (MSI-H), 12 (7%) showed low microsatellite instability (MSI-L), and 118 (67%) showed MSS. All MSI samples were deletion mutants and all 12 MSI-L cases had MSI in dinucleotide markers (D5S346, D2S123, and D17S250). MSI was significantly associated with proximal colon cancers ( p<0.0001), as previously reported. The MSI-H tumors were also associated with tumor node metastasis (TNM) I/II stages ( p=0.05) and high-grade tumors ( p<0.01). Here, we propose a DHPLC-based method as an alternative for MSI analysis.