A multiplex PCR assay consisting of 13 Rapidly Mutating Y STR loci called RM-Yplex was previously developed. Platinum (R) Taq DNA polymerase was used to amplify the 13 Y STR loci in a single reaction at an amplification time of approximately 2.5 h. In order to shorten the process with reliable results, two DNA polymerases were tested with the multiplex. Phusion (R) Flash High Fidelity, TAKARA Z-taq (TM), and Platinum (R) Taq DNA polymerases were investigated for conducting RM-Yplex assay at various PCR cycling conditions. Rapid, robust, and efficient amplification of all the markers within the multiplex were achieved. The amplification time was reduced from 2.5 h to less than 28 min with Phusion (R) Flash High Fidelity DNA polymerase using Veriti (R) PCR thermal cycler.