Using several clonal lineages of Simocephalus vetulus (Cladocera, Daphniidae) as a random sample, we investigated the genetic component of the halotolerance of one brackish and two freshwater populations of this littoral filter feeder. We hypothesized that genotypes from the brackish population were more tolerant than freshwater ones, via adaptation to local environmental conditions. Clonal identity was established by a cost-effective molecular fingerprinting technique (microsatellite-primed polymerase chain reaction (MSP-PCR)). Two distinct methodologies were used to assess cladoceran sensitivity to synthetic-grade sodium chloride (NaCl): (i) standard 48-h acute assays and (ii) 12-h survival time (ST) trials. No correlation was found between acute EC50 and ST values. The sensitivity of brackish and freshwater clones was comparable in terms of acute EC50 (varied from 2.28 to 3.83 g.L−1). On the contrary, genetically determined differential tolerance to NaCl among populations was found for ST: all brackish genotypes, except one, were more resilient (ST>120 min) than freshwater clones (ST<120 min). Bearing in mind that these results were obtained with isolates from the extant population, it is surprising that the range of acute sensitivity of the freshwater and brackish genotypes was similar, and that the only difference between them was the ability of brackish clones to survive longer under high salinity stress (6 g.L−1, in ST trials). We must conclude that the effect of salinity (original environment context) on the selection of genotypes was weaker than we had expected and than other authors have shown for other stressors.