Objective To develope a high throughput mutational detection method by mutiple fluorescence-labeled polyrnerase chain reaction (PCR) products. Methods A total of 27 known mutations including 22 substitutions, 3 iosertions (1, 2 and 7 bp) and 2 deletions (t and 2 bp) in the hepateeyte nuclear factor (HNF)-4a, glucokinase and HNF-la genes were tested. During nested PCR, amplified fragments were labeled with three fluorescent dyes. PCR products were visualized with an ABI-3T/ fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions. Results Twenty-five of 27 variants (93%) could be detected by combining 5% gtycerol and 10% sucrose gel matrix conditions. Twenty-two of 27 (82%) and 18 of 27 (67%) variants were identified using 5% glycerol and 10% sucrose conditions, respectively. Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple, non-hazardous, time-savino and sensitive method for hiah throuahout mutation detection.