Permethrin resistance in the human head louse, Pediculus capitis De Geer (Anopulura: Pediculidae), has been reported worldwide, is associated with the knockdown phenotype, and elicits cross-resistance to DDT and the pyrethrins. Two point mutations, T929I and L932F, in the voltage-sensitive sodium channel α-subunit gene are responsible for permethrin resistance as a resistant haplotype (kdr-like). We have optimized a serial invasive signal amplification reaction (SISAR) protocol for the detection of these mutations using PCR amplified DNA fragments. SISAR distinguished all genotypes with high accuracy in a head louse population from Texas that was heterogeneous in terms of permethrin sensitivity. Using SISAR, resistance-conferring mutations are detected in a high throughput format, facilitating the efficient monitoring of permethrin resistance allele frequency in field populations.