We report the results of typing three short tandem repeat (STR) polymorphisms by a multiplex system in three forensically relevant populations of Australia; (i) a general Australian, (ii) a Caucasian, and (iii) an Asian sample. The STRs are tetranucleotide repeats in the following autosomal genes; CSF1PO, TPOX and THO1, and collectively comprise the CTT multiplex (Promega). In all three population samples, each of the loci exhibited genotype frequencies consistent with those expected under Hardy-Weinberg equilibrium, and, further, there was no significant deviation from random association of alleles across separate loci. These two characteristics, plus the high discrimination power values, demonstrate that the CTT multiplex system is a powerful tool for DNA profiling in Australian jurisdictions. Australian Asians and Caucasians exhibit frequencies of alleles at these three STR loci very similar to those in other Asian and Caucasian samples. While most of the variation exists within any single population, there are still considerable differences between Asians and Caucasians. This difference between the two groups is minimal with respect to TPOX and greatest for THO1 alleles.