Brook troutSalvelinus fontinalisfrom 21 unstocked waters, 3 naturalized lakes, and 4 hatcheries in New York and Pennsylvania were analyzed electrophoretically for allozyme expression. Thirty-two of the 68 loci examined were polymorphic. Average heterozygosity of samples from populations classified as wild–unstocked was 0.050 (range, 0.026–0.076). Differences (P< 0.05) occurred among the 21 wild–unstocked samples at 25 of 31 possible locus comparisons. All wild–unstocked samples were significantly different from each other and from hatchery samples (P< 0.01). A high fixation index (FST= 0.375) indicated that the wild–unstocked samples represented highly differentiated populations. A considerable portion of the gene diversity was found among major river basins (22.5%); the remainder was due to differences among minor river drainages within basins (10.0%) and among samples within minor drainages (5.0%). Cluster analysis of genetic distances organized samples into three main groups that were also associated by river basins. Management strategies for conserving the genetic variability of wild brook trout should focus on individual lake and stream populations within river basins as primary management units. Data indicated that naturalization had varying success in preserving the gene pools of the progenitor populations.