Recently, the Yfiler(®) Plus PCR Amplification Kit (Yfiler(®) Plus, Thermo Fisher Scientific, Waltham, MA, USA) was introduced. Yfiler(®) Plus amplifies 27 Y-chromosomal short tandem repeat loci (Y-STRs) and adds ten new Y-STRs to those analysed with the commonly used AmpFlSTR(®) Yfiler(®) PCR Amplification Kit (Yfiler(®), Thermo Fisher Scientific, Waltham, MA, USA). Seven of the new Y-STRs are rapidly mutating Y-STRs (RM Y-STRs). In this study, 551 male individuals from Denmark, Greenland and Somalia were typed with Yfiler(®) Plus. The results were compared to those obtained with Yfiler(®) in the same individuals. Forensic and population genetic parameters were estimated for Yfiler(®) Plus. Yfiler(®) Plus had a higher power of discrimination than Yfiler(®) in all three populations. Compared to Yfiler(®), Yfiler(®) Plus offers increased power of discrimination, which is obviously an advantage in crime case investigations. However, the inclusion of seven RM Y-STRs in Yfiler(®) Plus makes it less attractive for relationship testing because of the relatively high combined mutation rate, approximately 15%.