Short tandem repeat (STR) typing is widely used in forensic investigation. When the same DNA sample is analyzed with different STR typing kits, a typing discrepancy is occasionally observed. In this study, we examined the cause of a typing discrepancy in a sample at D5S818 locus. This sample was designated as 10, 12 using Identifiler®, Identifiler® Plus, GlobalFiler®, PowerPlex® 16HS, and PowerPlex® 18D, but as 9.3, 12 using PowerPlex® Fusion. Sequencing results indicated that the shorter allele in the sample had a deletion (U31Tdel) at 31 nucleotides upstream of the repeat region (AGAT)10. This deletion was located in the binding site of the published D5S818 forward primer in PowerPlex® 16 and was only 9 and 11 nucleotides downstream of our estimated 5′ end position of D5S818 forward primer in GlobalFiler® and PowerPlex® 18D, respectively. We also examined the effect of primer length on the heterozygous peak balance in this sample.