Six Artemia populations from coastal and inland sites in Chile, ranging from 20 to 50 degrees latitude south, were compared morphologically. The study included reference samples of A. franciscana (San Fransisco Bay, California-USA) and A. persimilis (Buenos Aires, Argentina). These species are likely to be found in Chile. Samples from Peru (Piura) and Brazil (Macau, Rio Grande do Norte) are two known examples of deliberate introduction of A. franciscana. The hypothesis of A. franciscana being the dominant species in South America was tested by multivariate morphological analyses based on ten body measurements. In addition, laboratory cross-fertility tests were performed in order to evaluate levels of reproductive isolation among these widely distributed populations. The analysis showed that A. franciscana and A. persimilis are morphologically divergent. A number of populations overlap with the San Francisco Bay sample, two are morphologically close to Buenos Aires, whilst others lie morphologically in between. Interpopulation morphological differences along with geographical and, probably, ecological divergence do not reflect reproductive isolation, at least of the pre-mating type. The morphological similarity of two Chilean samples with A. persimilis suggests that the distribution of A. persimilis should be further investigated. A multi-trait approach for Artemia characterization is stressed as a way to obtain better descriptions and interpretations of the biological diversity in the genus.