Carbamate insecticides have been used for over four decades to control brown planthopper, Nilaparvata lugens, but resistance has been reported in many countries, including the Republic of Korea. The bioassay results on resistance to fenobucarb showed that the LC50 values were 3.08 for the susceptible strain, 10.06 for the 2015 strain, and 73.98 mg/L for the 2019 strain. Compared to the susceptible strain, the 2015 and 2019 strains exhibited resistance levels 3.27 and 24.02 times higher, respectively. To elucidate the reason for the varying levels of resistance to fenobucarb in these strains, mutations in the acetylcholinesterase 1 (ACE1) gene, the target gene of carbamate, were investigated, but no previously reported mutations were confirmed. Through RNA-seq analysis focusing on the expression of detoxification enzyme genes as an alternative resistance mechanism, it was found that the carboxylesterase gene Nl-EST1 was overexpressed 2.4 times in the 2015 strain and 4.7 times in the 2019 strain compared to the susceptible strain. This indicates a strong correlation between the level of resistance development in each strain and the expression level of Nl-EST1. Previously, Nl-EST1 was reported in an organophosphorus insecticide-resistant strain of Sri Lanka 2000. Thus, Nl-EST1 is crucial for developing resistance to organophosphorus and carbamate insecticides. Resistance-related genes such as Nl-EST1 could serve as expression markers for resistance diagnosis, and can apply to integrated resistance management of N. lugens.