A validation study was performed for the AmpFlSTR Yfiler PCR Amplification Kit (the Yfiler kit) to evaluate its potential applicability to Y-STR analysis in forensic casework. The optimal DNA input for PCR amplification was approximately 1 ng of male DNA. A marker-specific stutter ratio to filter stutter peaks higher than that defined by the kit manufacturer has been adopted for some markers to more readily and efficiently type Japanese male samples. No non-specific amplification was observed using 2000 ng female DNA as the PCR template. A full or partial DNA profile was detected from DNA mixture samples containing 1 ng of male DNA and 2000 ng of female DNA. From a mixture of two male DNA samples, all alleles possessed by the minor DNA contributor were detected when DNA derived from the minor contributor was present at 20% of the total DNA and above. Several loci were amplified when analyzing 23-year-old bloodstain samples derived from male individuals. Since they had not yet been defined by the kit manufacturer, we sequenced new alleles observed in Japanese males to assign accurate allele names. Analyses were also carried out of unusual alleles (an allele with poor amplification at DYS390 and apparent null alleles at DYS437 and DYS448) we observed in Japanese males. It is thought important that forensic biologists are aware of new and unusual alleles for DNA profile interpretation. Our validation study indicated the usefulness of the Yfiler kit for analyzing forensic biological samples containing male DNA.