The Yfiler (Ⓡ) Plus Amplification Kit amplifies 27 Y chromosomal small tandem repeat (STR) markers. The kit has five-fluorescent dye chemistry and the improved PCR buffer system of modern STR kits. We validated the kit for accredited investigations of crime scene samples by a thorough study of kit dynamics and performance. We determined dye-dependent analytical thresholds by receiver operating characteristics (ROC) and made a customised artefact filter that includes theoretical known artefacts by use of previously analysed population samples. Dilution series of known male DNA and a selection of crime scene samples were analysed with the customised thresholds and artefact filters. The Yfiler (Ⓡ) Plus Amplification Kit was sensitive giving full profiles down to 70 pg of male DNA. The balances between the fluorescent dyes as well as between loci were very good. The kit was able to produce full Y-STR profiles from crime scene samples containing small amounts of male DNA and large amounts of female DNA (although unspecific reactions were evident for very unbalanced mixtures). A decrease in the drop-out rate was found for both the dilution series and population samples, as well as a small increase in the drop-in rate for population samples, using the customised threshold and artefact filters compared to company-provided thresholds and artefact filters. The additional drop-ins were all of a nature that would be detected by inspection of the results. For the crime scene samples, large amounts of female DNA complicated the analysis by causing drop-ins of characteristic female DNA artefacts. Even though the customised analytical threshold in combination with the custom-made artefact filters gave more alleles, crime scene samples still needed special attention from the forensic geneticist.