This is a study on the diversity of entomopathogenic nematodes (EPN) in Chile and the bionomics of a new EPN species, Steinernema australe. Surveys for EPN were carried out in six regions of Chile, representing some of the principal habitats of the country and including desert, sub-polar, montane and rainforest habitats, by collecting approximately 1400 soil samples. Live-baiting of the samples revealed 101 isolates, 94 of which were Steinernema and seven were Heterorhabditis. Morphological and molecular characterisation revealed one species of Heterorhabditis, designated as Heterorhabditis sp. 1 and three species of Steinernema, viz., S. feltiae and two previously undescribed species, S. australe and S. unicornum. Heterorhabditis sp. 1 was only found in the northern half of the country, the majority of Steinernema were found in the southern half. The most southerly survey, Patagonia and Tierra del Fuego, had the highest proportion of positive soil samples at 18.3%, made up of S. feltiae and S. unicornum; surveys in the Atacama Desert had the lowest return (1-2%), consisting only of Heterorhabditis sp. 1. There were indications of molecular and geographical intraspecific variation in S. feltiae and S. unicornum. The incidence of the four species and a summary of habitat characteristics are provided, as well as taxonomic descriptions of S. australe, S. unicornum and Heterorhabditis sp. 1. Profiling of S. australe revealed a fast life- cycle, with new infective juveniles (IJ) observed after 6 days at 20°C and significant infection at cool, humid conditions. Five days at ea -1°C had no effect on IJ survival, but subsequent infectivity was reduced, a possible chilling-injury. Steinernema australe infected a wide range of insect pests, appearing most effective against Lepidoptera. No nictating or jumping behaviour was observed. Xenorhabdus sp. and Paenibacillus sp. of bacteria were recovered from S. australe.