A knowledge of genetic structure in natural populations is often necessary for conservation and management purposes, especially in declining Pacific salmon populations. To test for genetic differentiation between nine populations of chinook salmon, Oncorhynchus tshawytscha, from south-western British Columbia, Canada, DNA was extracted from 603 fish and hybridized with a single-locus minisatellite probe. Multivariate statistical analyses of the resulting allele size data permitted successful overall population identification of 52% (individual population range: 24–78%; P < 0.005), indicating a high level of genetic differentiation among the nine populations. Two of the nine populations were further analysed using data from a second minisatellite locus. The discrimination success rate improved from 81.1% (one-locus analyses) to 90.0% (two-locus analyses), indicating the potential for greatly increased resolution gained by the addition of more loci. These results indicate that variation at minisatellite loci can be used for assessing population-level genetic structure, even with artificial gene flow.