Weed species diversity may benefit from organic farming due to enhanced temporal diversification of crop species in a rotation and omission of herbicide applications. However, in intensively managed conventional systems, little evidence exists as to what extent diversified crop rotations contribute to higher weed species richness. Using an on-farm approach, the effect of crop rotation (organic, conventional diverse (CD) and conventional simple (CS) crop rotations) and weed control (with vs. without) on weed species richness, cover, community composition and crop biomass, was analysed in 24 winter wheat fields. Weed species with beneficial functions for invertebrates and birds were analysed separately. Weed species richness was higher in the organic crop rotation, but did not differ between CD and CS crop rotations. Weed control treatment reduced species richness in both conventional rotations, but not in the organic one. Redundancy analyses revealed that crop rotation intensity accounted for the largest part of the explained variation in weed species composition. Results from the study indicate that the maintenance of weed species richness and conservation of species with important ecological functions requires not only temporal diversification of crop species in the rotation, but also an adjustment of weed control strategies.