Genetic markers can be defined as the sequence of DNA that has a known physical location on the chromosome. Generally, markers help to link the inherited disease with the responsible gene. In forensics, Short Tandem Repeats (STRs) are most commonly used gold standard markers that consist of repeated 2-10 base pair units. Now days, in paternity cases 20 or more autosomal STR markers are using to establish paternity or other forensic application. It is reported in many cases one or two autosomal markers of the offspring's DNA profile show different alleles which are not present in their parent's DNA profile. This might be possible due to mutation or incest paternity cases. Therefore, to confirm mutation or support to establish paternity in such cases, lineage markers viz. Y-STRs, X-STRs, and Mt. DNA are potential tools. In this article, the characterization and forensic significance of X-STRs in mutated autosomal markers of two paternity cases were discussed. X-STRs of X-chromosomes show their unique pattern of inheritance than other autosomal STRs. The X chromosome is inherited in the offspring from father and mother both. The X chromosome is inherited from mother to daughter and son; and from father to daughter only. X-STR are used to decode genetic structure in respect to the cases like parentage, human identification, kinship, and disease identification. In this paper, significant role of X-chromosomal STR analysis in mutated autosomal STRs especially in paternity cases has been discussed.