BackgroundWaterhemp (Amaranthus tuberculatus(Moq.) Sauer) and Palmer amaranth (Amaranthus palmeriS. Wats.) are two dioecious and important weed species in the world that can rapidly evolve herbicide-resistance traits. Understanding these two species' dioecious and sex-determination mechanisms could open opportunities for new tools to control them. This study aims to identify the differential expression patterns between males and females inA. tuberculatusandA. palmeri. Multiple analyses, including differential expression, co-expression, and promoter analyses, used RNA-seq data from multiple tissue types to identify putative essential genes for sex determination in both dioecious species.ResultsGenes were identified as potential key players for sex determination inA. palmeri.GenesPPR247,WEX,andACD6were differentially expressed between the sexes and located at scaffold 20 within or near the male-specific Y (MSY) region. Multiple genes involved with flower development were co-expressed with these three genes. ForA. tuberculatus,no differentially expressed gene was identified within the MSY region; however, multiple autosomal class B and C genes were identified as differentially expressed and possible candidate genes.ConclusionsThis is the first study comparing the global expression profile between males and females in dioecious weedyAmaranthusspecies. Results narrow down putative essential genes for sex-determination inA. palmeriandA. tuberculatusand also strengthen the hypothesis of two different evolutionary events for dioecy within the genus.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04286-9.