A new Heterorhabditis species of entomopathogenic nematode was isolated from soil of the Atacama Desert in Chile. The new species is characterized by morphometrics of the infective juvenile (IJ) with length (L) = 611 (578–666) μm, head to excretory pore length (EP) = 115 (101–126) μm, tail = 69 (62–79) μm long, (EP/tail) × 100 (E%) = 165 (149–182) and L/maximum body diameter (ratio a) = 28 (25–31). The male has spicules 45 (40–49) μm long, gubernaculum 20 (17–22) μm long and (spicule length/anal body diameter) × 100 (SW%) = 205 (179–249). The hermaphroditic adult has shallow cuticular folds immediately anterior and posterior to the vulva, a slight post-anal swelling and a finely rounded tail terminus. Morphologically, H. atacamensis n. sp. resembles H. safricana, H. marelatus, H. downesi and H. amazonensis, but can be distinguished by characters of adult and IJ stages. In particular, for adult males, H. atacamensis n. sp. differs from H. amazonensis by the number and orientation of the genital papillae and from H. downesi by the position of the excretory pore; by the shape of the female tail terminus from H. downesi and by the position of the IJ hemizonid from H. marelatus. Heterorhabditis atacamensis n. sp. is further characterized by internal transcribed spacer (ITS) and D2D3 rDNA sequences, the closest species, H. safricana, being separated by 13 bp across 730 bp of the ITS (incorporating ITS1 (partial sequence), 5.8S (complete sequence), ITS2 (complete sequence)) and 5 bp across 592 bp of the partial 28S (incorporating D2D3) sequence. The morphological and molecular data confirm that H. atacamensis n. sp. is a valid species.