The dynamics of dissemination of the environmental human pathogen Vibrio parahaemolyticus are uncertain. The O3:K6 clone was restricted to Asia until its detection along the Peruvian coasts and in northern Chile in 1997 in phase with the arrival of El Niño waters. A subsequent emergence of O3:K6 strains was detected in austral Chile in 2004. The origin of these 1997 and 2004 population radiations has not yet been conclusively determined. Multiple loci VNTR analysis using seven polymorphic loci was carried out with a number of representative strains from Asia, Peru and Chile to determine their genetic characteristics and population structure. Asian and Chilean subpopulations were the most genetically distant groups with an intermediate subpopulation in Peru. Population structure inferred from a minimum-spanning tree and Bayesian analysis divided the populations into two genetically distinct groups, consistent with the epidemic dynamics of the O3:K6 clone in South America. One group comprised strains from the original Asiatic population and strains arriving in Peru and Chile in 1997. The second group included the remaining Peruvian Strains and Chilean strains obtained from Puerto Montt in 2004. The analysis of the arrival of the O3:K6 clone at the Pacific coasts of South America has provided novel insights linking the origin of the invasion in 1997 to Asian populations and describing the successful establishment of the O3:K6 populations, first in Peru and subsequently in the South of Chile owing to a possible radiation of Peruvian populations. © 2010 Blackwell Publishing Ltd.