BackgroundEntomopathogenic nematodes (EPNs) have emerged as promising biological control agents against various insect pests, with particular significance for location-specific applications. Native EPNs are especially valuable for controlling prevalent pests in their respective regions. The high-temperature semi-arid regions of India remain relatively unexplored in terms of the presence and potential of EPNs. This study represents the first comprehensive report on Steinernema pakistanense isolates from a semi-arid region, focusing on their isolation, identification, parasitic potential, and molecular characterization.ResultsA total of 240 soil samples were collected from agricultural fields in Rajasthan, India, where ten samples tested positive for EPN Steinernema pakistanense using the Galleria mellonella baiting technique. Morphological and morphometric analyses confirmed their classification within the "bicornutum" group of Steinernema species, with notable variations in body length and other parameters compared to the original type population. Genetic analysis using ITS and D2-D3 rDNA regions confirmed a high degree of similarity (99.73-100% for ITS and 99.76-99.88% for D2-D3) to S. pakistanense, validating their species identity. Phylogenetic analysis revealed close evolutionary relationships among the isolates and distinct divergence from other taxa within the genus. Toxicity assays demonstrated significant pathogenicity against both G. mellonella and Tenebrio molitor larvae, with isolates BSBT_S5, BSBT_S6, and BSBT_S9 exhibiting particularly high efficacy. Isolate BSBT_S10 was reported for its exceptional reproductive potential, producing over 1.8 million infective juveniles per larva of G. mellonella at optimal dosages.ConclusionChemical pesticide resistance leads to the urge to apply biocontrol agents such as (EPNs) with high pathogenicity and reproductivity. Semi-arid areas of India are unexplored and have the potential for the presence of EPNs that could be utilized in extreme environmental conditions. Morphological, molecular, and phylogenetic analysis revealed the identity of ten EPN isolates as S. pakistanense as the first study of its type in the semi-arid area of India. All isolates are proven highly toxic to the larvae of G. mellonella and T. molitor as representative of lepidopteran and coleopteran insects and reproduce in high numbers. The potential of isolated nematodes is anticipated in insect pest management.