Some troublesome weeds such as Ambrosia artemisiifolia have led farmers to adopt herbicide-tolerant varieties (HTVs) in cultivated sunflower fields. Agricultural practices associated with the use of HTVs have raised concerns among public authorities, prompting the recommendation to monitor the potential effects of HTVs on biodiversity. In this context, we surveyed the vegetation of 239 sunflower fields and their margins in three French regions between 2017 and 2019, with a specific focus on A. artemisiifolia. We collected information on 21 explanatory variables covering agricultural practices, landscape factors, and spatiotemporal data. Herbicide-tolerant varieties fields were associated with lower weed diversity but similar field margin community diversity. This lower weed diversity can be attributed to the greater use of herbicides and shorter crop rotations, whereas landscape factors may be more important for the diversity in field margins. Conventional fields with traditional varieties showed lower abundance of A. artemisiifolia compared with organic fields and HTV fields. A. artemisiifolia abundance was higher in the most infested region, in fields with a high proportion of sunflower crops in the crop rotation, late sowing dates, high numbers of hoeing operations, and high numbers of post-emergence treatments. We interpreted the association of hoeing and HTV use with a high abundance of A. artemisiifolia as a response to weed infestation rather than its cause. In conclusion, no unintended effects of HTVs were found in field margins, but practices associated with HTVs lead to lower weed community diversity and HTV fields had still high A. artemisiifolia abundance after weed control.