StR multiplex reactions are today standard in paternity testing. For more difficult constellations such as complex kinship analysis, paternity testing in deficiency cases or within related individuals the application of multiple brands of the commercial kits does not always provide the desired discriminatory power as the majority of the loci are shared. Therefore a new multiplex system for the resolution of cases of complex kinship analysis or deficiency cases based on multicolor dye technology was constructed. Nine loci (D10S2325, D12S391, Penta E, D7S1517, SE33, Penta D, D8S1132, D17S976, FGA), each with high discriminating power, were selected. We performed a population study and evaluated these loci. The statistical values of the multiplex reaction for discriminating power and power of exclusion were almost the same as in the commercial Powerplex 16-Kit. The usefulness of the reaction was shown. Three cases of paterity were solved by using the reaction. Microsatellite instability was observed by analyzing some tissues of thyroid carcinoma with the new multiplex reaction, so that the clinical use was demonstrated. This novel, multiplex polymerase chain reaction amplification and typing system for nine STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.