Field studies have established high potential for hybridization between two important and often coexisting weedy species, Amaranthus hybridus and Amaranthus tuberculatus. Prezygotic reproductive barriers between these species are believed to be limited to pollen competition and availability. A greenhouse study showed that a herbicide-resistance gene (ALS) from A. hybridus could be introgressed into an advanced A. tuberculatus background (BC2). However, evidence is lacking in support of such transfer in nature. Postzygotic reproductive barriers may minimize, if not preclude, natural introgression. Indeed, A. hybridus ×A. tuberculatus hybrids are characterized by reduced fertility and even floral neuterism. The purpose of this study was to assess hybrid fertility in the BC1 generation and its relationship with genome structure and segregation at ALS. Fertility was assessed by measuring seed output and by pollen evaluation, and segregation at ALS was determined via a molecular marker system. The two parental species have the same ploidy (2n = 32) but differ in DNA content (2C) values, with A. tuberculatus chromosomes being on average 29% greater than those of A. hybridus. Given that most (98%) BC1s were homoploid, 2C values were used as indicators of relative genomic constitution. Fertility in the BC1 generation was greater than that of F1s, and 3% of BC1s had seed output similar to that of the parental species. Fertility in the BC1 did not correlate (in a strict way) with reconstitution of parental genomes. Hybrid sterility appeared to be controlled by relatively few loci. Heterozygosity at ALS was negatively correlated with fertility. Also, the A. tuberculatus ALS allele was not observed in the A. hybridus sexual condition, monoecism. Linkage of ALS to a locus associated (directly or via epistasis) with hybrid sterility may explain the fertility penalty observed with ALS introgression. Moreover, this linkage might explain why sequenced herbicide-resistance ALS alleles from sympatric A. tuberculatus and A. hybridus populations show independent evolution.