Mysids are common and abundant organisms in estuaries and coastal waters, where they have a central role in the functioning of food webs. The Río de la Plata (RdlP) is a major estuary that affects biogeographic patterns of marine coastal organisms in the south-west Atlantic, but studies of the plankton in the RdlP are scarce. Knowledge of the mysid fauna in particular is very limited with a single species (likely alien, Neomysis americana), traditionally known to inhabit this 38,000 km2 estuary, and a second one, Mysidopsis tortonesei, only recently reported. The purpose of this paper is to contribute with the knowledge of the mysid fauna of this ecosystem by exploring the spatial distribution and in situ salinity preferences of N. americana and M. tortonesei in the mixohaline region of the RdlP during austral autumn, 2001. Both species showed clear and contrasting haline affinities that resulted in an almost perfect spatial segregation within the study area. Neomysis americana occurred preferentially in oligo- and meso-haline conditions (range <1 to 33.37, but most abundant at salinities <28), while M. tortonesei occurred at salinities >28. Temperature range was quite narrow and it was unlikely that it affected mysid distribution in the estuary. Temporal variability in observed patterns, as well as the role of biological interactions in shaping spatial distribution of N. americana and M. tortonesei within the estuary are subjects that deserve further attention in future studies.