Background <p><i>Culex tritaeniorhynchus</i> is the primary vector of Japanese encephalitis virus (JEV), a disease of global public health significance, and is widely distributed across Africa, Asia, Australia, and Europe. Despite its epidemiological importance as a JEV vector, studies on insecticide resistance in <i>Cx. tritaeniorhynchus</i> populations from the Republic of Korea (ROK) remain extremely limited. This study aimed to investigate the spatial and temporal occurrence of knockdown resistance (<i>kdr</i>) mutations in <i>Cx. tritaeniorhynchus</i> populations from the ROK. Additionally, the association between phenotypic resistance to pyrethroid insecticides and <i>kdr</i> alleles was examined.</p> Methods <p><i>Culex tritaeniorhynchus</i> specimens were collected from eight locations across the ROK to assess the occurrence of <i>kdr</i> mutations. Additionally, WHO tube bioassays were conducted using the pyrethroid insecticide deltamethrin to assess phenotypic resistance and its association with <i>kdr</i> mutations.</p> Results <p>The <i>kdr</i> mutation 1014F was detected in all surveyed populations, and the frequency of the 1014F allele remained relatively stable during the peak abundance period of <i>Cx. tritaeniorhynchus</i> from July to September. Bioassay results further indicated that <i>Cx. tritaeniorhynchus</i> populations in the ROK exhibit resistance to pyrethroid insecticides. However, no clear association was detected between the presence of the 1014F mutation and pyrethroid-resistant phenotypes.</p> Conclusions <p>These findings indicate the need for further investigations into insecticide resistance in <i>Cx. tritaeniorhynchus</i> to support effective vector control and mitigate the risk of JEV transmission. Moreover, the results suggest that pyrethroid resistance in <i>Cx. tritaeniorhynchus</i> may not be driven solely by <i>kdr</i> mutations, indicating the potential involvement of additional resistance mechanisms.</p>