The major branches of life diversified in the marine realm, and numerous taxa have since transitioned between marine and freshwaters. Previous studies have demonstrated higher rates of molecular evolution in crustaceans inhabiting continental saline habitats as compared with freshwaters, but it is unclear whether this trend is pervasive or whether it applies to the marine environment. We employ the phylogenetic comparative method to investigate relative molecular evolutionary rates between 148 pairs of marine or continental saline vs. freshwater lineages representing disparate eukaryote groups, including bony fish, elasmobranchs, cetaceans, crustaceans, mollusks, annelids, algae, and other eukaryotes, using available protein-coding and non-coding genes. Overall, we observed no consistent pattern in nucleotide substitution rates linked to habitat across all genes and taxa. However, we observed some trends of higher evolutionary rates within protein-coding genes in freshwater taxa—the comparisons mainly involving bony fish—compared with their marine relatives. The results suggest no systematic differences in substitution rate between marine and freshwater organisms.