Population-genetic and morphometric investigations were undertaken in 28 populations of the palaemonid prawn Macrobrachium nipponense with different-sized eggs which occur in different habitats from estuaries to inland waters of the Japanese islands, in order to elucidate the process of their differentiation. In estuarine populations with small eggs, allele frequencies in the Pgm locus changed with a cline along the Pacific coast. This appears to have resulted from infrequent unidirectional gene flow by sea current-mediated larval dispersal. Estuarine populations along the coast of the Sea of Japan, on the other hand, were genetically homogeneous, suggesting more frequent gene flow in them. Populations with medium and large eggs, which are observed in brackish- or fresh-water limnetic systems of sea-relict lake origin on both sides of the Japanese islands, are considered to have differentiated from estuarine populations after the Holocene marine transgression. Some lacustrine populations were significantly different in their genetic content and morphometric traits, implying their independent derivation.