The admixed Brazilian population shows high levels of genetic variability, which resulted from the contribution of three main ethnicities, Amerindian, European, and African. However, due to its huge territory, admixing has been asymmetrical, i.e., the relative contribution from each ethnicity has been unequal in the five geopolitical regions of the country. The aim of this study was to describe genetic variability using a panel of short-tandem repeats on the X chromosome (X-STR) in order to perform a comprehensive evaluation of the usefulness of such markers for forensic purposes in Brazil. Twelve X-STR (DXS9895, DXS7132, DXS6800, DXS9898, DXS6789, DXS7133, GATA172D05, DXS7130, HPRTB, GATA31E08, DXS7423, and DXS10011) were chosen and tested in a sample of 2,234 individuals belonging to 16 out of the 27 Brazilian States, representing all of its five geopolitical regions. No markers showed significant deviation from the Hardy-Weinberg equilibrium, even when analyses were partitioned to represent geopolitical regions. Genetic diversity per locus ranged from 67% (DSX7133) to 95% (DXS10011), and the State of Ceará showed the highest average genetic diversity (79% for all 12 X-STR markers). Considering the Brazilian population as a whole, the power of discrimination of the 12 X-STR panel in females (PDF) was 0.999999999999994, while the power of discrimination in males (PDM) was 0.9999999969. Such high values suggest the potential of that panel to be used in forensic applications and relatedness tests among individuals. Comparisons among the Brazilian populations investigated revealed significant differences when they were compared among each other, a pattern that was maintained when additional populations from Europe and Latin America were compared to Brazilians. Our results highlight the need and usefulness of specific genetic database for forensic purposes in Brazilian populations.