Entomopathogenic nematodes (EPNs) are important biocontrol agents, and accordingly, isolation and identification of native field-collected EPNs are required in order to successfully utilize for controlling endemic insect pests. The leopard moth, Zeuzera pyrina L. (Lepidoptera: Cossidae) is the most economically damaging pest in walnut trees in Iran with increasing importance in the last few years. In the present research, during a survey of entomopathogenic nematodes in various agricultural fields in Kerman region, several entomophilic nematodes were recovered from soil samples using the Galleria bait method. Based on preliminary tests on efficacy of recovered nematode isolates to re-infect the wax moth larvae and confirm Koch's postulates, after identifying one of the isolates based on classical and molecular studies, it was selected for further bioassay studies. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses using 18S, ITS and 28S region of ribosomal DNA identified this isolate as Acrobeloides maximus. Preliminary pathogenicity assessment of this indigenous isolate indicated that A. maximus was high virulent isolate against the larvae of Z. pyrina. Subsequently, the efficacy of A. maximus isolate against this insect pest was carried out in further experiments, including pathogenicity assays in plate and branch as well as evaluation of reproduction and penetration potential of this EPN. Plate assays were performed using a range of EPN concentrations (5, 10, 20, 50 and 100 infective juveniles (IJs) per larva) in laboratory. The LC50 value of A. maximus was 12.1 IJs larva-1 against Z. pyrina larvae after 72 h. This EPN caused high insect mortality in branch experiments and dissection of cadavers confirmed nematode infection. The results of the penetration and reproduction assays indicated that A. maximus was able to successfully penetrated and reproduced in the haemocoels of Z. pyrina and G. mellonella larvae. The highest reproduction was recorded at 20 IJs larva-1 in Z. pyrina (30560.5±559.3 IJs). In conclusion, our findings demonstrate that A. maximus, the first recorded isolate from Iran, is virulent to Z. pyrina larvae and it causes infection and successfully recycles in this pest. Notwithstanding the effectiveness of this pathogenic agent on Z. pyrina larvae, further studies are required to better track infection of treated larvae within the walnut tree trunks.