The personal identification of monozygotic (MZ) twins is of great importance in forensic medicine. Due to the extreme similarity in genetic between MZ twins, it is challenging to differentiate them using autosomal STR genotyping. Forensic experts are striving to explore available genetic markers that can differentiate between MZ twins. With the advent of next-generation sequence (NGS), an increasing number of genetic markers have been demonstrated to effectively differentiate between MZ twins. Here, we summarized for the relevant studies on MZ twins' differentiation and discussed the limitations of the underlying markers. In details, single-nucleotide variants (SNVs), copy number variation (CNV), mitochondrial DNA (mtDNA), DNA methylation, and non-coding RNA have been demonstrated considerable value. Furthermore, the utilization of proteomics, metabolomics, and microbiomics has shed light on MZ twin differentiation. Additionally, we introduce the methodologies for MZ differentiation based on external morphological variations observed in the human body. Looking to the future, the process of aging may represent a novel avenue for the differentiation of MZ twins.