The objectives of the study included the detection of genetic variation of 21 autosomal short tandem repeats (STRs) loci from random unrelated individuals in the middle and South Arab people of Iraq, the Forensic efficiency parameters of the autosomal 21 genetic loci using power plex21® kit and to evaluate the importance of these loci for forensic genetic purposes and the possibility to use the new kit in routine practical work. FTA® Technology was utilized to extract DNA from blood collected on FTA™ paper. Twenty one (21) STR loci including D3S1358, D13S317, PentaE, D16S539, D18S51, D2S1338, CSF1PO, Penta D, THO1, vWA, D21S11, D7S820, TPOX, D8S1179, FGA, D1S1656, D5S818, D6S1043, D12S391, D19S433 and Amelogenin were amplified by using power plex21® kit. PCR products were detected by genetic analyzer 3130xL then, the data processed and analyzed by PowerStatsV1.2 software. Several statistical parameters of genetic and forensic efficiencies based on allelic frequencies have been estimated. This includes the observed heterozygosity (Ho), expected heterozygosity (He), paternity index (PI), random match probability (RMP), power of discrimination (PD), chance of exclusion (CE), polymorphic information content (PIC) and P-value. The power of discrimination values for autosomal tested loci was from 75 to 96% therefore those loci can be safely used to establish a DNA-based database for Iraq population for identifical purpose. The high PIC values of the selected markers confirm their usefulness for genetic polymorphism studies and linkage mapping programs in human as well. The mean heterozygosity observed, expected and mean PIC values across the 20 loci were 0.79, 0.83 and 0.81, respectively indicating high gene diversity. A total of nine off-ladder alleles and one tri-allelic pattern were detected in this study.