Colonisation of freshwater habitats by marine animals is a remarkable evolutionary event that has enriched biodiversity in freshwater ecosystems. The acquisition of tolerance to hypotonic stress during early life stages is presumed to be essential for their successful freshwater colonisation, but very little empirical evidence has been obtained to support this idea. This study aimed to comprehend the evolutionary changes in osmoregulatory mechanisms that enhance larval freshwater tolerance in amphidromous fishes, which typically spend their larval period in marine (ancestral) habitats and the rest of their life history stages in freshwater (derived) habitats. We compared the life history patterns and changes in larval survivorship and gene expression depending on salinity among three congeneric marine-originated amphidromous goby species (Gymnogobius), which had been suggested to differ in their larval dependence on freshwater habitats. An otolith microchemical analysis and laboratory-rearing experiment confirmed the presence of freshwater residents only in G. urotaenia and higher larval survivorship of this species in the freshwater condition than in the obligate amphidromous G. petschiliensis and G. opperiens. Larval whole-body transcriptome analysis revealed that G. urotaenia from both amphidromous and freshwater-resident populations exhibited the greatest differences in expression levels of several osmoregulatory genes, including aqp3, which is critical for water discharge from their body during early fish development. The present results consistently support the importance of enhanced freshwater tolerance and osmoregulatory plasticity in larval fish to establish freshwater forms, and further identified key candidate genes for larval freshwater adaptation and colonisation in the goby group.