DNA fragment analysis, based on amplification of intersimple sequence repeats by the polymerase chain reaction (ISSR-PCR), was used to assess genetic relationships of 217 U.S. accessions of common cocklebur. Twenty-four polymorphic markers were generated from six primers. Analysis of genetic similarity by clustering procedures resulted in separation of the accessions into two main clusters. Accessions within these two clusters were designated as either northern or southern genotypes. Forty-four of 48 accessions analyzed from Washington, Michigan, Iowa, and Ohio were northern genotypes, whereas 67 of 68 accessions analyzed from Mississippi, Arkansas, South Carolina, and North Carolina were southern genotypes. Accessions from Kansas, Missouri, and Illinois included 40 and 56 northern and southern genotypes, respectively, indicating a transition zone. In Illinois, accessions collected from northern and southern counties tended to be northern and southern genotypes, respectively. We conclude that much of the genetic variation among U.S. common cocklebur accessions is distributed along a latitudinal gradient.Nomenclature:Common cocklebur, Xanthium strumarium L. XANST.