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A total of 150 samples of unrelated males from North of Italy were analyzed with the prototype Yfiler® Plus kit (Thermo Fisher Scientific, Oyster Point, CA). This kit is a short tandem repeat (STR) assay based on six-dye chemistry that amplifies 25 Y-STR loci. Sixteen loci are in common with Yfiler® kit and nine are new (DYS576, DYS627, DYS460, DYS518, DYS570, DYS449, DYS481, DYF387S1a/b, DYS533). In this population study, the improvement of adding additional Y-STR markers increased the discriminatory capacity from 0.787 with the minimum haplotype (MHT) loci to 1 with the prototype Yfiler® Plus kit.