Pollen-mediated gene flow (PMGF) might play an important role in dispersing herbicide resistance alleles in dioecious weedy Amaranthus species. Field experiments in a concentric donor–receptor design were conducted to quantify two sets of PMGF studies, an interspecific (Amaranthus tuberculatus 3 Amaranthus palmeri) and an intraspecific (A. tuberculatus 3 A. tuberculatus). In both studies, PMGF was evaluated using a resistant A. tuberculatus phenotype with enhanced mesotrione detoxification via P450 enzymes as a source of resistance alleles. For interspecific hybridization, more than 104 000 putative hybrid seedlings were screened with three markers, one phenotypic and two molecular. The two molecular markers used, including 2-bp polymorphisms in the internal transcribed spacer region, distinguished A. palmeri, A. tuberculatus and their hybrids. Results showed that 0.1% hybridization between A. tuberculatus 3 A. palmeri occurred under field research conditions. For intraspecific hybridization, 22 582 seedlings were screened to assess the frequency of gene flow. The frequency of gene flow (FGF) varied with distance, direction and year of the study. The farthest distance for 90% reduction of FGF was at 69 m in 2015 however, after averaging across directions it was 13.1 and 26.1 m in 2014 and 2015, respectively. This study highlights the transfer of metabolism-based mesotrione resistance from A. tuberculatus to A. palmeri under field research conditions. The results presented here might aid in the rapid detection of A. palmeri among other Amaranthus species and show that PMFG could be expediting the increase of herbicide resistance in A. palmeri and A. tuberculatus across US crop production areas.