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Y-chromosomal short tandem repeats (Y-STRs) are essential to relate male lineages in forensic and evolutionary studies. Although large panels of Y-STR markers are now available, none possess sufficient discrimination power to distinguish close male relatives. This limit may be overcome by the use of rapidly mutating Y-STRs (RM Y-STRs), characterized by mutation rates higher than common Y-STRs. Recently, multicenter studies evaluated the ability of RM Y-STRs to differentiate father–son pairs; however, more extensive data on distantly related males are needed.A total of 157 male relative pairs separated by two to seven meiotic events, originating from Italy, were analyzed by 13 RM Y-STRs and 23 Y-STRs.Overall, 154 mutational events were observed at RM Y-STR loci and the estimated mutation rate was of 2.59 × 10−2 (95% confidence interval, 2.16 × 10−2−2.97 × 10−2). A total of 105 male relative pairs showed at least one mutation in at least one locus and differentiation rates increased from 52.8% to 88.9% from the second to the fourth generation, while 23 Y-STRs provided much lower values, spanning from 10.1% to 29.6%.These findings confirmed the higher capability of RM Y-STRs than conventional Y-STRs to resolve male lineages, thus suggesting a possible future use for forensic male individual identification.