This study establishes deltamethrin resistance in a common bed bug, Cimex lectularius L., population collected from New York City (NY-BB). The NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared with an insecticide-susceptible population collected in Florida (FL-BB). General esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase activities of NY-BB were not statistically different from those of FL-BB. cDNA fragments that encoded the open reading frame of voltage-sensitive sodium channel α-subunit genes from the FL-BB and NY-BB populations, respectively, were obtained by homology probing polymerase chain reaction (PCR) and sequenced. Sequence alignment of the internal and 5′ and 3′ rapid amplification of cDNA ends (RACE) fragments generated a 6,500-bp cDNA sequence contig, which was composed of a 6,084-bp open reading frame (ORF) encoding 2,027 amino acid residues and 186-bp 5′ and 230-bp 3′ untranslated regions (5′ and 3′ UTRs, respectively). Sequence comparisons of the open reading frames of the α-subunit genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly (Bemisia tabaci). V419L, located in the IS6 transmembrane segment, is a novel mutation. A Val to Met mutation at the corresponding position of the bed bug V419, however, has been identified in the tobacco budworm as a kdr-type mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB through a knockdown-type nerve insensitivity mechanism.