The ontogeny of osmoregulatory organs was studied in two geographically isolated populations of the palaemonid shrimp Macrobrachium amazonicum, one originating from the Amazon estuary (A) and the other from inland waters of the Pantanal (P) in northeastern and southwestern Brazil, respectively. A previous investigation had shown that the estuarine population is able to hypo-osmoregulate in seawater, whereas the hololimnetic inland population has lost this physiological function. In the present study, the structural development of the branchial chamber and excretory glands and the presence of Na+/K+-ATPase (NKA) were compared between populations and between larval and juvenile stages after exposure to two salinities representing hypo- and hypertonic environments. In the newly hatched zoea I stage of both populations, gills were absent and NKA was localized along the inner epithelium of the branchiostegite. In intermediate (zoea V) and late larval stages (decapodids), significant differences between the two populations were observed in gill development and NKA expression. In juveniles, NKA was detected in the gills and branchiostegite, with no differences between populations. At all developmental stages and in both populations, NKA was present in the antennal glands upon hatching. The strong hypo-osmoregulatory capacity of the early developmental stages in population A could be linked to ion transport along the inner side of the branchiostegite; this seemed to be absent or weak in population P. The presence of fully functional gills expressing NKA appears to be essential for efficient hyper-osmoregulation in late developmental stages during successful freshwater adaptation and colonization.