Ambrosia artemisiifolia and Ambrosia trifida (Asteraceae) are important pest species and the two greatest sources of aeroallergens globally. Here we took advantage of a hybrid to simplify genome assembly and present high quality chromosome level assemblies for both species. These assemblies show high levels completeness with BUSCOs of 94.5% for A. artemisiifolia and 96.1% for A. trifida and LTR Assembly Index values of 26.6 and 23.6, respectively. The genomes were annotated using RNA data identifying 41,642 genes in A. artemisiifolia and 50,203 in A. trifida. More than half the genome is comprised of repetitive elements, with 62% in A. artemisiifolia and 69% in A. trifida, and this is similar to other species in the Heliantheae alliance . Single copies of herbicide resistance associated genes PPX2L, HPPD, and ALS where found, while 2 copies of the EPSPS gene were identified; this latter observation may reveal a possible mechanism of resistance to the herbicide glyphosate. The evolution of genome structure has differed among these two Ambrosia species. The genome of A. trifida has undergone greater rearrangement, possibly the result of cataclysmic reorganization through chromoplexy. In contrast, the genome of A. artemisiifolia retains a structure that makes the allotetraploidization of the most recent common ancestor of the Heliantheae alliance the clearest feature of its genome. When compared other Heliantheae alliance species, this allowed us to reconstruct the common ancestor’s karyotype – a key step for further development of our understanding of the evolution and diversification of this economically and allergenically important group.