Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.