Entomopathogenic nematodes (EPN) are useful biological control agents of insect pests. However, the infective juvenile (IJ) stage which is the only stage to occur outside the host is susceptible to environmental extremes such as desiccation. We have isolated desiccation-tolerant strains of the EPN Heterorhabditis megidis. In this paper we describe the surface properties of these desiccation-tolerant mutants. Heterorhabditid IJs retain the sheath of the previous larval stage. The mutant lines possess alterations in the surface properties of the sheath. Differences were observed in fluorescent lipid analogue insertion into the surface of the sheath. Furthermore, cationized ferritin-binding studies demonstrated that the mutant lines possessed an increase in net negative surface charge. Removal of the surface layer of the sheath resulted in the loss of the mutant phenotype and in a reduction in the desiccation tolerance of the parental strain. Therefore, the negatively charged ‘surface coat’ appears to play an important role in the desiccation tolerance of Heterorhabditis species.