The infestation with the human ectoparasite, Pediculus humanus capitis (De Geer), is a common public health problem affecting schoolchildren worldwide. In Chile, the main active ingredients present in the over-the-counter pediculicides contain pyrethroids. Despite the extended use of these products, there is no evidence of the insecticide resistance status of the head lice geographically located in Chile. The most extended resistant mechanism of pyrethroids consists of the target site insensitivity (Kdr) determined by the presence of mutations linked to insecticide-binding sites in the voltage-sensitive sodium channel. T917I is recognized as the main mutation in head lice, and detection is considered to be a biomarker of resistance. The goal of the present study was to detect the presence and distribution of T917I mutation in five geographic locations of Chile. All five geographically selected louse populations had a frequency of pyrethroid resistance genes that ranged from 36 to 77%, and 94.9% of the collected head lice had one or two T917I mutant alleles. Moreover, the frequency of the aggregate resistant alleles was 50.5%. This is the first evidence that head lice in Chile had the mutations commonly associated with the resistance to pyrethroids. Moreover, the overrepresentation of heterozygotes in the studied populations suggests that head lice in Chile are currently under active selective pressure.