Esterase-5 is a highly polymorphic enzyme in Drosophila pseudoobscura and its close relatives. Numerous alleles have been identified by employing a number of physicochemical properties of the enzyme (e.g. electrophoretic mobility, enzyme stability, subunit dimerization, and variation in monomer–dimer equilibrium). Variation in the monomer–dimer equilibrium of esterase-5 leads to differences in electrophoretic mobility of monomers produced by dimers all of which have the same mobility. In this report we have used this criterion to study variation within, as well as between, four closely related species: D. pseudoobscura pseudoobscura, D. pseudoobscura bogotana. D. persimilis, and D. miranda. All lines were characterized for esterase-5 monomer and dimer mobility at a number of gel concentrations and the comparison was made by plotting log10 monomer – dimer mobility as a function of gel concentration. No variation was found within D. p. pseudoobscura or D. p. bogotana but some variation (two distinct alleles) did occur in D. persimilis. Drosophila miranda is segregating for two alleles, one of which is fixed in D. pseudoobscura and the other one is common in D. persimilis. Thus it seems that the variation in monomer–dimer equilibrium is a rather conservative criterion and that the variation in D. miranda is a good deal older than the speciation event(s) which gave rise to D. miranda and the lineage leading to D. pseudoobscura and D. persimilis.Key words: esterase-5, Drosophila pseudoobscura, monomer–dimer equilibrium, population, polymorphism, speciation, electrophoresis.