The application of large numbers of entomopathogenic nematodes (EPN) to control insect pests of agriculture is likely to have an impact on the local EPN fauna, yet little is known about the intraspecific relationships between EPN populations, particularly with regard to phylogeny and outbreeding. Here we assess the fitness, with regards to fecundity, host insect mortality and time taken to produce progeny, of isolates of Steinernema feltiae from Bull Island, Ireland. Exon-primed, intron-crossing (EPIC) PCR was used to examine intraspecific phylogenies between S. feltiae isolates, and identified up to three possible colonisation events of Bull Island. EPIC-PCR grouped two isolates, 33.D.(2) and 59.F.(2), separately from the remaining ten S. feltiae isolates These same two isolates consistently performed poorly in all fitness assessments. Following the crossbreeding of all isolates in Galleria mellonella, the number of host cadavers exhibiting emerging infective juveniles was significantly fewer than expected and there were significant differences between isolates in the number of days until progeny were observed. Host insect mortality varied between 40 and 87%. Such intraspecific variation may be a result of adaptation to different microhabitats of Bull Island, which in turn may be accentuated by laboratory culture practices.