Aconitum noveboracense, a rare, herbaceous perennial, is restricted to recently unglaciated areas in Iowa, Wisconsin, Ohio, and New York, and federally classified as a threatened species. These populations may be disjuncts of the common congener, A. columbianum Nutt., which occurs in the mountains of the western United States. Morphological characters do not reliably separate these taxa. The identity of Black Hills populations, located between the ranges of the rare and common species, is also uncertain. We characterized genetic variation within and among the Aconitum populations in question using isozymes and randomly amplified polymorphic DNA (RAPDs). Isozymes indicate a high degree of similarity among all populations and a high level of genetic diversity in Black Hills populations. Of 97 scorable RAPD loci, 89.7% are polymorphic and clearly resolve most populations. Like isozymes, RAPDs indicate high levels of genetic diversity in the Black Hills and very strong similarity of these populations to A. columbianum from the Bighorn Mountains. Aconitum noveboracense populations show >80% similarity to A. columbianum populations. A population of A. uncinatum from Ohio shows the greatest differentiation from other populations. Therefore, both isozyme and RAPD data concur with the recent treatment of A. noveboracense and A. columbianum as a single species.