The brine shrimp Artemia (Crustacea, Branchiopoda), a paradigmatic inhabitant of hypersaline lakes, has molecular features to survive under stressful conditions, such as the p26 heat shock protein. We report the RFLP fingerprinting pattern (four restriction enzymes) of a 217 bp fragment of exon2 of the Hsp26 gene in six Artemia franciscana and four Artemia persimilis populations, the most genetically divergent Artemia species co‐occurring in latitudinal extremes of Chile. The species‐specific RFLP pattern observed is a simple and cost‐effective single‐locus tool for species delimitation and experimental testing the habitat‐induced isolation barrier between them.