This study reported a sexual assault case where Y-STR DNA profile was tested for the confirmation of male DNA. Within the DNA profile of the sample of accused, a tri-allelic pattern was detected at locus DYF387S1. Since this locus doesn't exist in any of the commercially available Y-STR amplification kits, So, the confirmation of this tri-allelic pattern was done by re-extracting and amplifying the extracted DNA by using Y-Filer Plus amplification kit (Thermo Fischer Scientific, CA, USA-Thermo) followed by separation of amplicons on Genetic Analyzer 3500xl.The allelic patterns of case sample belong to Type-2 pattern of tri-allelic outcome because peak heights were in balanced manner. This region is very much prone towards any structural mutation which could be either addition or deletion of allele, so for the forensic point of view this locus should be used carefully for kinship analysis.