Protein and enzyme variation in 2 populations of the mud crabMacrophthalmus hirtipes(Jacquinot, 1853) (Ocypodidae) from 1 marine and 1 estuarine habitat were investigated by poly‐acrylamide gel electrophoresis. A survey of 24 loci revealed that 22 were common to both populations; 20 were monomorphic and 2 were highly polymorphic. Two alleles were detected for each of the polymorphic loci, esterase‐2 (EST‐2) and ester‐ase‐3 (EST‐3). The frequencies of the EST‐3 alleles were similar in the 2 populations. However, the frequencies of the 2 EST‐2 alleles in the estuarine population were significantly different from those for the marine population. Expression of 1 locus, esterase‐4 (EST‐4) was confined to the estuarine population. Two alkaline phosphatase loci (AKPH‐1 and AKPH‐2) were detected in the estuarine population, but only AKPH‐1 was found in the marine population. EST‐4 and AKPH‐2 were neither sex nor age specific. These interpopulational genetic differences may reflect differences in environmental conditions measured between the 2 habitats.