Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rondônia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus
Heterorhabditis
. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as
Heterorhabditis indica
, whereas LPP7 was considered
Heterorhabditis baujardi
. Comparative analysis of the ITS1 sequence of
H. indica and
H. baujardi isolates showed a polymorphic site for the restriction enzyme
Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as
H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as
H. baujardi.
