Objective The aim of this study was to investigate the effect of D5S818 locus allele losson paternity by PowerPlex21 system. Methods Chelex ⁃100 method was used to extract DNA from the samples.Multiplex amplifications of 20 autosomal STR loci were performed in 3248 families using PowerPlex21 system. Thegenotyping of STR loci was analyzed after capillary electrophoresis. The possible allelic loss at D5S818 locus wasverified by amplification by Identifiler system. Results Loss of allele 12 at D5S818 locus were found in sevenindividuals from five pedigrees using PowerPlex21 system and confirmed by Identifiler system. Conclusion Lossof allele was often misinterpreted as“mutation”of the locus，which has some influence on the result of combinedparentage index. For the accurate paternity testing result，different detection systems with the same STR locuscould be used.