In this study, the GlobalFiler Express amplification kit was evaluated for forensic use in 541 South African individuals belonging to the Afrikaaner, amaXhosa,1 amaZulu,1 Asian Indian and Coloured population groups. Allelic frequencies, genetic diversity parameters and forensic informative metrics were calculated for each population. A total of 301 alleles were observed ranging between 5 and 44.2 repeat units, 43 were rarely observed partial repeats and seven were novel. The combined match probability (CMP) ranged from 2.21x10 (Coloured) to 5.21x10 (AmaZulu), and the combined power of exclusion (CPE) 0.9999999978 (Afrikaaner) to 0.99999999979 (AmaZulu) respectively. No significant departures from Hardy-Weinberg equilibrium (HWE) were observed after Bonferroni correction. Strong evidence of genetic structure was detected using the coancestry coefficient? Analysis of Molecular Variance (AMOVA) and an unsupervised Bayesian clustering method (STRUCTURE). The efficiency of assignment of individuals to population groups was evaluated by applying likelihood ratios with WHICHRUN, and the individual ancestral membership probabilities inferred by STRUCTURE. Likelihood ratios performed the best in the assignment of individuals to population groups. Signs of positive selection were detected for TH01 and D13S317 and purifying/balancing selection for locus SE33. These three loci also displayed the largest informativeness for assignment (In) values. The results of this study supports the use of the GlobalFiler STR profiling kit for forensic applications in South Africa with the additional capability to predict ethnicity or continental origin of a random sample.