Nine hundred eighteen DNA-confirmed father-son pairs were typed for 16 Y-chromosomal short tandem repeat (Y-STR) markers by AmpFLSTR® Yfiler™ PCR Amplification kit. In a total of 15,606 allele transmissions, 36 mutations were detected. The average mutation rate across all 16 Y-STR markers was 0.0023 (95 % confidence interval, 0.0016-0.0032). One two-step mutation was found at DYS389II, and all other mutations were single steps. The losses and gains were balanced at all other loci, excluding DYS385 and DYS458, where losses were more frequent than gains. Mutation rates among different Y-STR loci were significantly different (Χ (2) = 69.05, P = 0.000). Mutation rates were correlated with the lengths of the alleles. Alleles with higher number of repeats were more likely to mutate. Mutation rates were also correlated with the gene diversity of the locus (r (2) = 0.565, P = 0.023). Loci with higher gene diversity had higher mutation rates. In addition, the mutation rate of the older father was found to be notably higher than that of the younger father.