ABSTRACT:This study aimed to detect mutations associated with pyrethroid resistance mechanism in different populations of Varroa destructor from Uruguay and Argentina with different degree of susceptibility to this family of acaricides. Bioassays were performed to evaluate the degree of susceptibility to flumethrin and tau-fuvalinate; then mites were studied in order to determine the presence or absence of the resistance-associated mutations by sequencing a region of the sodium channel gene. In the mites studied from Colonia, which showed resistance to flumethrin and susceptibility to tau-fluvalinate in the bioassays, L925V mutation was observed in heterozygous and homozygous state. The presence of the wild-type allele in homozygous state was identified in all mites from Treinta y Tres, in which acaricides have never been used. The mite population from Mar del Plata through bioassays were determined as susceptible to both pyrethroids, however, genotyping results showed the presence of heterozygous individuals (925 V/L) as well as homozygous wild type (925L), while homozygous resistant individuals (925V) were present at low frequency. This would suggest that this population presents a great potential to give a generalized resistance to pyrethroids. This study represents the first report of a L925V mutation in resistant mites to flumethrin from South America.