Differential resistance to metal pollution in Daphnia longispina populations was reported in previous studies. In this work, we tried to determine if variation in polymorphic enzymes, often referred as being under metal selection, were related with differences in resistance to acute single- and mixed-metal exposure. Allozyme genotype of 20 putatively polymorphic enzymes, 48-h median lethal concentration (LC50) for copper, and median lethal time (LT50) for a 3% dilution of acid mine drainage (AMD) were determined for 24 lineages of D. longispina. The copper LC50s ranged from 29.3 to 226 μg/L, and the AMD LT50s ranged from 48 min to 25 h and 29 min, with a strong correlation between both end points. Five distinct multilocus genotypes were identified based on polymorphisms in glucose-6-phosphate isomerase, lactate dehydrogenase, malate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP+), phosphoglucomutase, and peroxidase. No differences were found in average genotype sensitivity for both toxicity end points or in genotype frequencies between the resistant- and sensitive-lineage groups. The results obtained indicate that allozyme genotype is not associated with increased resistance to acute metal stress in D. longispina.