Closely related species are key models to investigate mechanisms leading to reproductive isolation and early stages of diversification, also at the genomic level. The brittle star cryptic species complex Ophioderma longicauda encompasses the sympatric broadcast- spawning species C3 and the internal brooding species C5. Here, we used de novo transcriptome sequencing and assembly in two closely related species displaying contrasting reproductive modes to compare their genetic diversity and to investigate the role of natural selection in reproductive isolation. We reconstructed 20 146 and 22 123 genes for C3 and C5, respectively, and characterized a set of 12 229 orthologs. Genetic diversity was 1.5-2 times higher in C3 compared to C5, confirming that species with low parental investment display higher levels of genetic diversity. Forty-eight genes were the targets of positive diversifying selection during the evolution of the two species. Notably, two genes (NHE and TetraKCNG) are sperm-specific ion channels involved in sperm motility. Ancestral sequence reconstructions show that natural selection targeted the two genes in the brooding species. This may result from an adaptation to the novel environmental conditions surrounding sperm in the brooding species, either directly affecting sperm or via an increase in male/female conflict. This phenomenon could have promoted prezygotic reproductive isolation between C3 and C5. Finally, the sperm receptors to egg chemoattractants differed between C3 and C5 in the ligand-binding region. We propose that mechanisms of species-specific gamete recognition in brittle stars occur during sperm chemotaxis (sperm attraction towards the eggs), contrary to other marine invertebrates where prezygotic barriers to interspecific hybridization typically occur before sperm–egg fusion.