A polymerase chain reaction (PCR)-based assay was used for the specific detection of Habronema microstoma and Habronema muscae (Nematoda, Spirurida) in order to identify the intermediate hosts of both nematode species under field conditions. A total of 1087 netted and 165 laboratory-bred flies were tested. Flies were identified as Musca domestica Linnaeus 1758, Musca autumnalis De Geer 1776, Haematobia irritans (Linnaeus 1758), Haematobia titillans (De Geer 1907) and Stomoxys calcitrans (Linnaeus 1758) (Muscidae). Genomic DNA was extracted from pools of fly heads, thoraces and abdomens, and 703 samples were subjected to a duplex two-step semi-nested PCR assay to specifically detect diagnostic regions within the ribosomal ITS2 sequence of both H. microstoma and H. muscae. Stomoxys calcitrans specimens were positive for H. microstoma DNA and M. domestica specimens were positive for H. muscae DNA. In particular, PCR-positive samples derived from both farm-netted and laboratory-bred flies. The present study represents the first evidence of the vectorial competence of different fly species as intermediate hosts of Habronema stomachworms under field conditions. We discuss the roles of S. calcitrans and M. domestica in transmitting H. microstoma and H. muscae.