Allelic frequencies for six polymorphic allozyme loci (27 loci examined), as determined by starch-gel electrophoresis, were not significantly different for alewife, Alosa pseudoharengus, among the Great Lakes (Ontario, Erie, Huron, and Michigan). Alewife from one of the Finger Lakes (Cayuga Lake) had allelic frequencies similar to Great Lakes alewife, and the alleles of Great Lakes alewife form a subset of those found in Cayuga Lake. In contrast, Maritime Canada populations (Gaspereau, Miramichi, and St. John rivers) were distinct from each other and from the Great Lakes and Cayuga Lake populations. Highly significant allelic frequency differences were observed among the Maritime populations and between the Maritime populations and the Great Lakes or Cayuga Lake populations. Fifteen alleles were not shared between the Great Lakes and the Maritime populations. We concluded from these results that Great Lakes alewife and Cayuga Lake alewife form a contiguous stock and that Great Lakes alewife are a recent invader that probably originated from the Hudson–Mohawk rivers via the Erie Canal and the New York Finger Lakes rather than the previously suggested alternative origin from the Canadian Maritimes via the St. Lawrence River.