This study evaluates the forensic utility of 23 autosomal short tandem repeat markers in 400 samples from the Kuwaiti population, of which four markers (D10S1248, D22S1045, D2S441 and SE33) are reported for the first time for Kuwait. All the markers were shown to exhibit no deviation from Hardy–Weinberg equilibrium, nor any linkage disequilibrium between and within loci, indicating that these loci are inherited independently, and their allele frequencies can be used to estimate match probabilities in the Kuwaiti population. The low combined match probability of 7.37 × 10–30 and the high paternity indices generated by these loci demonstrate the usefulness of the PowerPlex Fusion 6C kit for human identification in this population, as well as to strengthen the power of paternity testing. Off-ladder alleles were seen at several loci, and these were identified by examining their underlying nucleotide sequences. Principal component analysis (PCA) and STRUCTURE showed no genetic structure within the Kuwaiti population. However, PCA revealed a correlation between geographic and genetic distance. Finally, phylogenetic trees demonstrated a close relationship between Kuwaitis and Middle Easterners at a global level, and a recent common ancestry for Kuwait with its northern neighbours of Iraq and Iran, at a regional level.