The PowerPlex Y23 System is a 23-loci, 5-color Y-STR multiplex designed for genotyping forensic casework samples, database samples and paternity samples. The kit contains: all 12 loci in the current PowerPlex Y System, the additional 5 loci found in AmpFlSTR Y-filer, plus 6 new loci. An internal validation study of the PowerPlex Y23 kit was therefore conducted including the following aspects: sensitivity, mixture studies of male–male and female–male DNA, performance with simulated inhibition and stutter calculations. 100 Caucasians living in Switzerland were also typed using the PowerPlex Y23 kit.