Knotweed species in the genus Reynoutria are native to eastern Asia but have become noxious weeds in Europe and North America. In the United States, invasive populations of Japanese knotweed (Reynoutria japonica Houtt.), giant knotweed [Reynoutria sachalinensis (F. Schmidt) Nakai], and their interspecific hybrid known as Bohemian knotweed (R. × bohemica Chrtek & Chrtková) continue to expand their ranges. Although these plants are among the most invasive terrestrial species, there are relatively few molecular tools for identifying the parental species, the F1 hybrid, or subsequent hybrids or introgressed individuals. We studied Reynoutria populations in Wisconsin, a state where all three taxa grow, to determine whether molecular data would be useful for distinguishing species and identifying hybrids. We obtained DNA sequence data from the plastid matK gene and the nuclear LEAFY gene and compared these to previously published sequences. Data from the uniparentally inherited matK region included haplotypes attributable to R. japonica and R. sachalinensis. Nuclear data indicated that R. sachalinensis plants are most similar to native plants in Japan, with two Wisconsin accessions exhibiting a monomorphic genotype for the LEAFY gene. Three Wisconsin accessions of R. japonica were each characterized by having three distinct kinds of LEAFY sequence. Most plants in our study were found to possess two or three phylogenetically distinct copies of the LEAFY gene, with the copies being most closely related to R. japonica and R. sachalinensis, respectively, and these were inferred to be interspecific hybrids. Altogether, five kinds of interspecific hybrids were identified, reflecting various combinations of LEAFY sequence types from the parental species. The widespread existence of hybrid plants in Wisconsin, many of which are morphologically identifiable as R. japonica, indicates a cryptic genetic diversity that should be examined more broadly in North America using molecular tools.