While weed management is consistently a top priority among farmers, there is also growing concern for the conservation of biodiversity. Maintaining diverse weed communities below bioeconomic thresholds may provide ecosystem services for the crop and the surrounding ecosystem. This study was conducted to determine if weed diversity, density and biomass differ within and among organic and conventional crop rotations. In 2007 and 2008, we sampled weed communities in four long-term crop rotations near Mead, Nebraska using seedbank analyses (elutriation and greenhouse emergence) and above-ground biomass sampling. Two conventional crop rotations consisted of a corn (Zea mays) or sorghum (Sorghum bicolor)–soybean (Glycine max)–sorghum or corn–soybean sequence and a diversified corn or sorghum–sorghum or corn–soybean–wheat (Triticum aestivum) sequence. Two organic rotations consisted of an animal manure-based soybean–corn or sorghum–soybean–wheat sequence and a green manure-based alfalfa (Medicago sativa)–alfalfa–corn or sorghum–wheat sequence. Species diversity of the weed seedbank and the above-ground weed community, as determined by the Shannon diversity index, were greatest in the organic green manure rotation. Averaged across all sampling methods and years, the weed diversity index of the organic green manure rotation was 1.07, followed by the organic animal manure (0.78), diversified conventional (0.76) and conventional (0.66) rotations. The broadleaf weed seedbank density in the tillage layer of the organic animal manure rotation was 1.4×, 3.1× and 5.1× greater than the organic green manure, diversified conventional and conventional rotations, respectively. The grass weed seedbank density in the tillage layer of the organic green manure rotation was 2.0×, 6.1× and 6.4× greater than the organic animal manure, diversified conventional and conventional rotations, respectively. The above-ground weed biomass was generally greatest in the organic rotations. The broadleaf weed biomass in sorghum and wheat did not differ between organic and conventional rotations (CRs), but grass weed biomass was greater in organic compared to CRs for all crops. The above-ground weed biomass did not differ within CRs, and within organic rotations the grass weed biomass was generally greatest in the organic green manure rotation. The weed seedbank and above-ground weed communities that have accumulated in these rotations throughout the experiment suggest a need for greater management in long-term organic rotations that primarily include annual crops. However, results suggest that including a perennial forage crop in organic rotations may reduce broadleaf weed seedbank populations and increase weed diversity.