Abstract A new entomopathogenic nematode, Steinernema colombiense n. sp., is described from Colombia. Morphological, molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. In addition, 28S and ITS rDNA sequence data were used to assess evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. colombiense n. sp. include morphometric features of the third-stage infective juvenile, including body length of 636 (549-732) μm, narrow body diam. (31 (22-36) μm), position of the excretory pore (35 (31-40) μm), tail length (41 (32-53) μm), D% = 29 (25-33) and E% = 205 (138-284). In addition, males of first and second generations are characterised by the morphology of the spicules and gubernaculum, the number and arrangement of the genital papillae and the excretory pore position (at 67 (56-76) and 54 (46-63) μm, for first and second generations, respectively). In addition to these traits, 28S and ITS rDNA sequences analyses both showed this species to be a distinct and unique entity.