Kochia is an annual broadleaf weed species. Glyphosate resistant (GR) kochia has become a severe problem since the introduction of GR crops. In order to minimize the selection pressure of herbicides, IWM practices need to be involved to maintain sustainable cropping systems. Previous studies mainly investigated the IWM practices effects on kochia density or biomass in the field, but very limited studies have investigated the IWM practices effects on the copy number of the target gene of the gene for glyphosate resistance (EPSPS). To determine whether there were effects of IWM practices on kochia EPSPS copy number, a rotation experiment with wheat, soybean, corn, and canola in rotation with four replicates was conducted. Each replicate contained 16 plots with four treatments for each crop, including 1) a wide row spacing with low seeding rate treatment, 2) a wide row spacing with high seedling rate treatment, 3) a narrow row spacing with low seeding rate treatment, and 4) a narrow row spacing with high seeding rate treatment. The narrow row spacing treatment together with high seeding density can be an effective IWM practice to reduce the selection pressure for herbicide resistant kochia, but the effect of these treatments on EPSPS copy number are not known. In corn and soybean, the wide row spacing together with high seeding density IWM practice contributed to increased EPSPS copy numbers in 2018 which suggests that the biotic stress applied by the IWM treatment contributed to resistance evolution in that year.