Electrophoretic analysis of isozymes and mitochondrial DNA (mtDNA) restriction analysis were used to study the genetic divergence between the Acadian whitefish, Coregonus huntsmani, and members of the subgenera Coregonus (lake whitefish, C. clupeaformis) and Leucichthys (Arctic cisco, C. autumnalis, and lake cisco, C. artedii). Results obtained from both studies demonstrated that the Acadian whitefish is genetically highly distinct from the other coregonines examined. mtDNA restriction analysis revealed that the Acadian whitefish possesses a unique mitochondrial genotype which is divergent from that of the two cisco species or lake whitefish. Twelve of 13 restriction enzymes used were informative in distinguishing the Acadian whitefish from the other species, and species-specific fragment patterns were observed for 10 enzymes. In isozyme analysis of five loci, the Acadian whitefish was monomorphic at two loci for alleles not found in lake whitefish. Acadian whitefish also possessed an additional isozyme at another locus that was not found in lake whitefish and Arctic cisco specimens. This isozyme is unknown from the genetic model for lake whitefish at this locus. These results provided useful genetic markers to identify the Acadian whitefish. They emphasize that the extinction of the species would represent a major loss of both genetic diversity and potential information concerning the contentious phylogeny of coregonine fishes.