Currently, capillary electrophoresis-based Y-STR genotyping kits can detect up to 40 Y-STR loci simultaneously. However, these kits only report length-based genotypes and are unable to provide STR sequence information. The STRSeqTyperY68 kit, designed for forensic male pedigree differentiation using next-generation sequencing technology, excels at genotyping 67 Y-STR loci plus a sex-determinant locus in a single-tube reaction on the MiSeq FGx sequencing platform. It simultaneously provides both length and sequence polymorphism genetic information, simplifies testing procedures, enhances efficiency, and facilitates precise differentiation of male family lineages. The ITO method is commonly used to calculate the kinship index of two individuals’ biological relationship based on Mendel’s law of genetic segregation. Additionally, it evaluates consanguineous relationships within five degrees of kinship between two individuals. The combination of next-generation sequencing technology and the ITO method can effectively narrow down the range of potential families. This paper documents a rape and murder case that remained unsolved for eight years. By cooperatively utilizing the STRSeqTyperY68 kit and the ITO method, the potential connection between crime scene evidence and reference samples was evaluated. Gradually, the investigative leads were narrowed down, leading to the resolution of the case.