Thousands of polymorphic microsatellite loci are now mapped in the human genome, most of which exhibit a large number of segregating alleles. For forensic, evolutionary and gene mapping applications, it is important to establish intralocus allelic independence at these loci. We develop a test for intralocus allelic independence based on repeat size differences between alleles within genotypes of individuals, and show its relationship with the intraclass correlation of allele sizes within loci. Applications of this test to 13 short tandem repeat loci in 4 subpopulations indicate that the distribution of size differences of alleles has adequate power to detect intralocus dependence of alleles caused by population substructure. In addition, size differences between randomly chosen alleles provide information regarding heterozygosity and contiguity of allele sizes at the locus, which by themselves do not necessarily indicate the presence of population substructure.