Analysis of natural populations of Trypanosoma brucei has shown that there is linkage disequilibrium between alleles at pairs of loci in isolates taken from the field. This disequilibrium can occur as a result of a low frequency of genetic exchange, the masking of frequent genetic exchange by the rapid expansion of a few genotypes or by the treatment of 2 (or more) genetically isolated populations as a single population. We have analysed stocks from 2 geographically separate locations using 3 minisatellite markers to determine the frequencies of the alleles in each area and the frequency and nature of the multilocus genotypes. The results show that many alleles and multilocus genotypes are unique to each geographical location, supporting the conclusion that these populations are genetically isolated with limited or no gene flow between them. This geographical substructuring needs to be taken into account in considering the origins of the linkage disequilibrium in a number of populations.