Rhynchium brunneum is a widely distributed wasp species in South Eastern Asia. R. brunneum females were collected from rural provinces of Cambodia, and their total RNA and venom were extracted on site. To search for novel substances in venom, a subtracted cDNA library specific to the venom gland and sac was constructed. A total of 1118 expressed sequenced sequence tags (ESTs) were sequenced and assembled into 349 contigs (107 multiple sequences and 242 singletons). In this result, we found the putative neurotoxin (DTX protein precursor), antimicrobial peptides (teratocyte-specific caboxylesterase) together with typical major components of wasp venom (venom hyaluronidase, arginine kinase, phospholipase A2, serine/theonine protein phosphatase). Additional in-depth annotation would be required for further characterization of many unidentified genes found in the EST library.