DNA samples of 523 unrelated anonymized individuals (307 males and 216 females) born and living in the Czech Republic were genotyped using Investigator (R) Argus X-12 system in the following loci localized in four linkage groups: DXS10148, DXS10135, DXS8378, DXS7132, DXS10079, DXS10074, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134, DXS742. Haplotype frequencies were calculated for each LG (Linkage Group). The frequency of most common haplotype was 0.016, 0.036, 0.042, and 0.023 for LG1, LG2, LG3, and LG4, respectively. The combined power of discrimination was more than 0.999999999 both for female and male samples. The mean exclusion chance was 0.99999999 (trios) and 0.999999 (duos). Informativity and suitability of Investigator (R) Argus X-12 for kinship determination was assessed by computing in several female-female duos using LR (Likelihood Ratio) determination for autosomal STR (PowerPlex ESI-17), linked (Investigator (R) Argus X-12 system), and unlinked (X-STR Decaplex) X-STR kits. Investigator (R) Argus X-12 proved to be very useful for sibship determination, since its LR values were relatively similar to LR for autosomal STR kit. This work presents the first population data for Investigator (R) Argus X-12 system in the Czech Republic.