Y-chromosomal short tandem repeats (Y-STRs) at rapidly mutating (RM) loci have been suggested as tools for differentiating paternally related males. RMplex is a recently developed system that incorporates 26 RM loci and four fast-mutating (FM) loci, targeting 44 male-specific loci. Here, we evaluated the RMplex by estimating Y-STR mutation rates and the overall differentiation rates for 542 Korean father-son pairs, as well as the genetic population values for 409 unrelated males. RMplex performed well, distinguishing 50.7 % of the father-son pairs by at least one mutation, a value 10 times higher than the previously reported differentiation rate achieved using the PowerPlex® Y23 System. Of the 369 mutations, 361 (97.8 %) were single-step mutations, with locus-specific mutation rates varying from 1.8 × 10