Entomopathogenic nematodes (EPNs) from the Heterorhabditidae and Steinernematidae families are well-known biocontrol agents against numerous insect pests. The infective juveniles (IJs) are naturally occurring in the soil and their success in locating and penetrating the host will be affected by extrinsic/intrinsic factors that modulate their foraging behavior. Characterizing key traits in the infection dynamics of EPNs is critical for establishing differentiating species abilities to complete their life cycles and hence, their long-term persistence, in different habitats. We hypothesized that phenotypic variation in traits related to infection dynamics might occur in populations belonging to the same species. To assess these intraspecific differences, we evaluated the infection dynamics of 14 populations of Steinernema feltiae in two experiments measuring penetration and migration in sand column. Intraspecific variability was observed in the percentage larval mortality, time to kill the insect, penetration rate, and sex-ratio in both experiments (P < 0.01). Larval mortality and nematode penetration percentage were lower in migration experiments than in penetration ones in most of the cases. The sex-ratio was significantly biased toward female-development dominance (P < 0.05). When the populations were grouped by habitat of recovery (natural areas, crop edge, and agricultural groves), nematodes isolated in natural areas exhibited less larval mortality and penetration rates than those from some types of agricultural associated soils, suggesting a possible effect of the habitat on the phenotypic plasticity. This study reinforces the importance of considering intraspecific variability when general biological and ecological questions are addressed using EPNs.