A multiplex PCR assay consisting of 13 Rapidly Mutating Y STR loci called RM-Yplex was previously developed. Platinum® 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® Flash High Fidelity, TAKARA Z-taqTM, and Platinum® 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® Flash High Fidelity DNA polymerase using Veriti® PCR thermal cycler.