Canada thistle is a noxious weed that occurs in a wide range of habitats and is difficult to control because of its extensive root system and prolific seed production. Here, we focused on estimating the level of genetic diversity between populations in North Dakota as a first step in examining diversity across North America. Two types of genetic marker, intersimple sequence repeats (ISSRs) and microsatellites were used. Both marker types resulted in polymorphic alleles suitable for assessing diversity. Analysis of molecular variance (AMOVA), molecular diversity analyses, and cluster analysis were conducted. Highly significant variation was detected between populations (P < 0.01). The greatest variance recovered was between individuals within populations. Gene flow among populations in the Northern Great Plains was indicated by the presence of shared alleles between the North Dakota and Minnesota populations and in cluster formation. Multiple introductions and continued gene flow between populations has led to the continued success of Canada thistle as an invasive plant in North America.