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A systematic programme of surveys for entomopathogenic nematodes (EPN) was done in Chile between 2006 and 2008. The survey spanned the principal ecosystems of mainland Chile as well as a number of islands, and covered a wide range of habitats including the Atacama Desert, Andean Altiplano, temperate rainforests and subpolar territory. Nearly 1400 soil samples were collected, of which 7% were positive for EPN. Of 101 EPN isolates obtained, 94 were Steinemema spp. and seven were Heterorhabditis sp. Of the 94 Steinernema isolates, 39 were identified as Steinernema feltiae, the remainder being distributed between two new species, S. unicomum (52 records) and S. australe (three records). The Heterorhabditis isolates, all designated as Heterorhabditis sp.1, are referred to herein as H. cf. safricana. Steinemema feltiae and S. Unicornum were collected predominately in the south of Chile and were obtained from a range of habitats, including forests, open grassland, montane soils and coastal zones; neither species was recovered from the far north of the country (viz., desert soils in the Norte Grande region). Steinernema australe was found in only three soil samples, all from humid, cool, coastal localities in the south. Heterorhabditis cf. safricana was recovered from the northern regions, with most isolates found in or on the periphery of the Atacama Desert; they were not recovered from cooler, more humid regions of southern Chile. Molecular information indicated there were two subgroups of both S. unicomum and S. feltiae, with a geographical, intraspecific split of subgroups between the most southerly and the more central survey zones. All isolates were collected by ex situ baiting with waxmoth larvae and the natural hosts are unknown.