BACKGROUND: The analysis of X-chromosome markers can be valuable in particular situations, for example, deficiency kinship cases, where the putative father cannot be typed. X-chromosome short-tandem repeats (X-STRs) are widely used in forensic genetics, while the use of X-chromosome single-nucleotide polymorphisms (X-SNPs) is still limited. STUDY DESIGN AND METHODS: The forensic usefulness of a set of 25 SNPs located across the X-chromosome was analyzed in 13 populations. The applicability of the 25 X-SNPs in kinship testing was illustrated in two immigration cases where the conclusions based on the analysis of 15 autosomal STRs were ambiguous and, in one of the cases, misleading. The samples in the two cases were also typed for eight X-STRs, 52 autosomal SNPs, and seven autosomal variable number of tandem repeats. RESULTS: The combined power of discrimination of the 25 X-chromosome markers varied from one in 1.7 x 10(6) males to one in 7.8 x 10(9) females and the combined mean chance of exclusion varied between 99.78% in duos to 99.998% in trios. In the two immigration cases, the use of X-linked markers increased the number of genetic inconsistencies and the cases could be solved. The usefulness of X-chromosome markers was particularly illustrative in Case 1, where the typing of 25 X-SNPs would have been sufficient to exclude paternity. CONCLUSION: The high level of polymorphism, low degree of linkage disequilibrium, and very low probability of mutation of the 25 X-SNPs makes this set of markers suitable as a supplementary set of markers in relationship testing.