Complex kinship identification such as half-sibling identification is a difficult task in forensic biology Here we represented an approach in dealing with half-sisters from different mothers, with the combination of autosomal and X chromosomal short-tandem repeats (STRs) data. X chromosomal STRs can offer additional information, especially in some cases where autosomal STRs alone may not provide enough information for an accurate opinion. In this case, half-sister or unrelated relationship between two women (S1 and S2) with different mothers were distinguished. 23 autosomal and 31 X chromosomal STRs of S1, S2, S1's mother (M1), S2's mother (M2) and S1's grandmother (G1) were profiled with three different commercial kits. As to X-chromosome STRs, likelihood ratios (LRs) were calculated by FamLinkX with consideration of linkage, linkage disequilibrium, and mutations. When only the profiles of the two individuals (S1 and S2) were available, LRs between S1 and S2 were 1.1110 × 102 based on 23 autosomal STRs and 3.2257 om107 based on 31 X chromosomal STRs. When the maternal genotypes were taken into consideration, LRs increased to 2.5297 × 103 and 3.0563 × 1018. Therefore, both the DNA profiles of each mothers and X chromosomal STRs are important in dealing with the identification of half-sisters from different mothers.