The entomoparasitic nematode (EPN) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) is a promising candidate for the biological control of larvae of the maize pest Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). An easily applicable and economically feasible method is the inundative release of EPNs together with sowing of maize in spring. At that time, however, D. v. virgifera eggs are still diapausing and larval hatch will only start about four to 6 weeks after EPN application, depending on climatic conditions. Efficacy of the nematode application against the targeted pest species therefore depends on the ability of the biocontrol agent to persist in the pest's environment, at least until emergence of the first larvae. To address this key issue, 18 field trials were carried out at six locations in Germany, Austria and Hungary between 2010 and 2011. Fields differed in their soil type and in the presence or absence of a host. Nematodes were either applied as fluid stream (suspended in water) or as microgranules (without water) into the soil during the sowing process. Persistence of the nematodes was determined by examining soil samples, using larvae of Tenebrio molitor (Coleoptera: Tenebrionidae) as a bait. Results showed that EPNs, either applied as a suspension or as microgranules, were able to persist in the soil of all field sites. EPNs could be detected for at least 6 weeks in the soil. Persistence levels were higher in sandy soil types than in clay or silty soils. The applied EPN formulation had no effect on the persistence level of EPNs in the soil, and the presence or absence of the host insects had a minor effect. It can be concluded that EPNs applied with sowing of maize persist long enough to potentially control the later hatching larvae of the maize pest D. v. virgifera.