Sexual assault (SA) is a crime of violence against a person's body resulting in a physical trauma, mental anguish, and suffering for victims generating expenses for government intended criminal investigation, medical care, and psychological attention. During the crime scene investigation, the identification and recovery of biological evidence (BE) are utmost important, since sometimes these are the only way to prove sexual contact and the perpetrator's identity. The examiner, with the help of specific technologies and techniques, must be able to find evidence that otherwise could go unnoticed. Forensic laboratories identify biological evidence with systemized protocols and use molecular methods to generate DNA profiles based on the amplification and DNA sequencing. Before the arrival of the new-generation sequencers, the application of other markers (single nucleotide polymorphisms (SNPs), insertion-deletion of nucleotides (INDEL), or microhaplotypes (MHs)) was laborious, expensive, and not very informative for forensic purposes; however, now they are useful in this field. Next-generation sequencing (NGS) brought a new series of applications like epigenetics, microbiota, messenger RNA, and microRNA analysis and the inferences in the ancestry and phenotyping of individuals. In the end, the results obtained from such analyses and stored in databases are very useful for the identification of sexual aggressors.