Yfiler® Plus PCR Amplification Kit using a new 6-dye system detects 27 Y-STR loci. This paper describes our validation results of the Yfiler® Plus kit for forensic casework analysis. We examined sizing precision, baseline noise, sensitivity, stutter ratios, peak height ratios, spectral pull-up, the tolerance to PCR inhibitors, ability to obtain results from aged bloodstains and mixed DNA samples, and artifact peaks. In this study, electrophoresis was performed by using a 3500xL Genetic Analyzer and a 3130xl Genetic Analyzer. Data from 3500xL were analyzed with a threshold value of 175 RFU and data from 3130xl were analyzed with a threshold value of 150 RFU by using GeneMapper® ID-X software v1.4. AmpFℓSTR® Yfiler® PCR Amplification Kit was also examined in the sensitivity study, the aged bloodstains study and the inhibition study for comparison between the two Y-STR kits. In the sensitivity study, the Yfiler® Plus kit with the 3500xL generated full Y-STR profiles constantly from DNA amounts greater than or equal to 0.25 ng. Low input DNA amounts (≤0.125 ng) occasionally resulted in partial profiles. No full Y-STR profile was obtained from less than or equal to 0.031 ng DNA amounts. In the spectral pull-up study, the 3130xl showed higher spectral pull-up ratios in use of Yfiler® Plus kit than the 3500xL. In the inhibition study, in comparison with the conventional Yfiler® kit, the Yfiler® Plus kit showed higher tolerance toward hematin and humic acid. In the mixture study, full Y-STR profiles were obtained from 1 ng of male DNA mixed with 4000 ng of female DNA. Our validation studies indicate that the Yfiler® Plus kit can be used for Y-STR analysis in forensic casework.