Phosphine, a highly sought-after fumigant, is utilized to combat insect pests infesting bulk-stored food grains. However, sustainability concerns arise as many stored grain pests are developing resistance to this compound worldwide. This study aimed to assess the extent of phosphine resistance in 6 north Indian populations of the lesser grain borer, Rhyzopertha dominica, collected from storage units where phosphine is regularly employed for pest control. In south India, this species has been reported to exhibit strong phosphine resistance. Resistance to phosphine typically arises due to genetic variations at the rph2 locus, which influence the activity of the DLD gene. Protein model was generated and employed to forecast the structure of DLD (Dihydrolipoamide dehydrogenase) proteins discovered in R. dominica. The structure from the selected populations revealed the presence of a characterized phosphine variant ‘P49S’ in most of the phosphine resistant populations. The characterized key mutation P49S occupy the FAD binding site in the DLD protein. Besides, amino acid mutations- A55T, E69K, E106, V114G, D119Y, and N158H also occupy the FAD biding site and V225G occupies the NADH binding sites. These findings would help further researchers to expedite the phosphine resistance management strategies.