Colonisation of freshwater habitats by marine animals is a remarkable evolutionary event that has enriched biodiversity in freshwater ecosystems. For successful freshwater colonisation, high physiological plasticity is presumed to be necessary, but its evolutionary basis has not been detailed. Marine-originated amphidromous species, which regularly migrate between freshwater and marine environments, have repeatedly lost migratory behaviour in many lineages, which sometimes triggered species radiation in freshwater habitats. Since amphidromous species typically visit the sea during the larval period, the difficulty in the evolution of larval freshwater tolerance is a bottleneck for freshwater colonisation. To elucidate the key evolutionary changes that enhance the physiological plasticity for freshwater colonisation, we compared larval gene expression changes depending on salinity conditions among three congeneric amphidromous goby species (Gymnogobius) with varying dependences on freshwater habitats. First, an otolith microchemical analysis and rearing experiment under laboratory conditions confirmed the presence of freshwater residents only in G. urotaenia and higher larval survivorship of this species both in seawater and freshwater conditions than the obligate amphidromous G. petschiliensis and G. opperiens. Larval whole-body transcriptome analysis revealed that G. urotaenia exhibited the greatest differences in the expression levels of several osmoregulatory genes, including aqp3, which is critical for water discharge from their body during early fish development. Thus, we obtained the results that consistently support the importance of enhanced osmoregulatory plasticity for establishing freshwater forms, and further identified some important evolutionary changes for larval freshwater adaptation and colonisation in the goby group.