In this study, we report a three-step mutation at D8S1132 locus between an alleged father (AF) and child in a paternity case. Three autosomal STR multiplex amplification kits were used for capillary electrophoresis detection. At the D8S1132 locus, the genotypes were: alleged father 17/23, parent mother 22, and child 20/22, which does not comply with Mendelian inheritance laws. Paternity was confirmed by calculating the cumulative paternity index and a three-step mutation was identified at the D8S1132 locus. Next-generation sequencing was further used to validate the experimental results and explore the source of the mutation. The results indicated that the allele 20 of the child was derived from the allele 23 of the alleged father. In addition, next-generation sequencing platform that simultaneously detected different genetic markers such as STR, SNP, and mitochondria DNA increased the cumulative parental index, which further confirmed the paternal relationship. Therefore, this case suggests that in paternity testing, when multiple mutations occur at a certain STR locus, capillary electrophoresis and next-generation sequencing can be combined for cross-validation to improve the credibility of the identification results.