Abstract: The male-specific Y-chromosomal short tandem repeat (STR) is a useful tool in forensic casework. The Y haplotype comprised of 16 loci, which is amplified simultaneously by AmpFlSTR® YfilerTM PCR kit and provides strong exculpatory evidence in individual identification. We reported a rare Y-STR profile with a null allele at the DYS448 locus and an off-ladder allele at the DYS456 locus, when genotyping material from a vaginal swab in an alleged rape case. Sequence analysis revealed that the DYS448 null allele was a true type of null allele because of a total deletion of 11 upstream repeats and 9 bp of the N42 region, and there were numerous primer binding site mutations as well. The amplicon of the DYS456 locus was a small 92-bp fragment that was off-ladder, and sequencing analysis showed that there were only 10 repeats (AGAT)10. This Y chromosome haplotype that was comprised of two variations provided helpful evidence for personal identification.