Elucidating the molecular basis of adaptation to different environmental conditions is important because adaptive ability of a species can shape its distribution, influence speciation, and also drive a variety of evolutionary processes. For crustaceans, colonization of freshwater habitats has significantly impacted diversity, but the molecular basis of this process is poorly understood. In the current study, we examined three prawn species from the genus Macrobrachium (M. australiense, M. tolmerum, and M. novaehollandiae) to better understand the molecular basis of freshwater adaptation using a comparative transcriptomics approach. Each of these species naturally inhabit environments with different salinity levels; here, we exposed them to the same experimental salinity conditions (0‰ and 15‰), to compare expression patterns of candidate genes that previously have been shown to influence phenotypic traits associated with freshwater adaptation (e.g., genes associated with osmoregulation). Differential gene expression analysis revealed 876, 861, and 925 differentially expressed transcripts under the two salinities for M. australiense, M. tolmerum, and M. novaehollandiae, respectively. Of these, 16 were found to be unannotated novel transcripts and may be taxonomically restricted or orphan genes. Functional enrichment and molecular pathway mapping revealed 13 functionally enriched categories and 11 enriched molecular pathways that were common to the three Macrobrachium species. Pattern of selection analysis revealed 26 genes with signatures of positive selection among pairwise species comparisons. Overall, our results indicate that the same key genes and similar molecular pathways are likely to be involved with freshwater adaptation widely across this decapod group; with nonoverlapping sets of genes showing differential expression (mainly osmoregulatory genes) and signatures of positive selection (genes involved with different life history traits).