Allozyme variation was characterised by starch gel electrophoresis at 23 enzyme coding loci and one regulatory locus in Atlantic salmon from 53 rivers in Eastern Canada, encompassing the majority of the species' North American range. Variation among rivers was highly heterogeneous and eight of the 15 polymorphisms showed regionally restricted distributions. Nearest neighbour joining (NJ) analysis and multi‐dimensional scaling suggest six distinct regional groups; Labrador/Ungava, Gulf of Saint Lawrence, Newfoundland (excluding Gulf rivers), the Atlantic shore/Southern Uplands of Nova Scotia, the inner Bay of Fundy, and the outer Bay of Fundy. Approximately 25% of observed genetic variation was distributed among these regions with a weak though significant overall correlation of genetic and geographic distance (Mantel Test, r = 0·255, P = 0·005). Collectively, the rivers showed consistent divergence from European populations with strong bootstrap support for the two clusters across loci in the NJ analysis. Mean heterozygosity was 0·061 for both continental groups, but the European population showed more than twice the variation among populations. FST values were 0·076 and 0·176 for North America and Europe, respectively, with an overall FST of 0·330.