Biennial wormwood is native to North America and has become an important weed problem in soybean and dry bean fields of North Dakota, South Dakota, and Minnesota in the United States and in the prairie provinces of Canada. Intersimple sequence repeat (ISSR) markers were used to study the genetic diversity among six biennial wormwood and one annual wormwood populations. Deoxyribonucleic acid (DNA) sequences from internal transcribed spacer (ITS1 and ITS2) regions of ribosomal DNA and morphological diversity among the biennial and annual wormwood populations were also studied. High levels of genetic diversity were evident with Nei's gene diversity statistic (h) = 0.40 for biennial wormwood and h = 0.36 for annual wormwood. Total diversity of six biennial wormwood populations was H T = 0.40, and 22% of this diversity was among populations (G ST = 0.22). Estimated gene flow among biennial wormwood populations was low (Nm = 0.9), and high levels of differentiation may be due in part to low levels of genetic exchange among biennial wormwood populations. Although biennial wormwood behaves more like an annual than a biennial, the ISSR, ITS, and morphological studies show that the two species are dissimilar.