Varroa destructor is a major parasitic mite of the Western honey bee (Apis mellifera) responsible for severe colony losses. Control of Varroa relies heavily on synthetic acaricides such as tau-fluvalinate, a pyrethroid that acts on the voltage-gated sodium channel (VGSC) of mites. However, repeated use of tau-fluvalinate has led to the evolution of resistance, jeopardizing treatment efficacy. This study aimed to assess the relationship between an in vitro phenotypic bioassay for taufluvalinate susceptibility and a molecular assay targeting the L925V mutation in the VGSC, a known marker of pyrethroid resistance. The objective was to evaluate the reliability of the L925V mutation as a predictive indicator of phenotypic resistance across multiple French Varroa populations. Thirteen Varroa destructor populations were sampled from treated and untreated colonies in France. Susceptibility to taufluvalinate was assessed through an in vitro phenotypic assay quantifying mite mortality following exposure to the active substance (at the lethal concentration for 90% of susceptible mites). Following exposure, mites were classified as susceptible (dead or immobile) or resistant (surviving). In parallel, a TaqMan allelic discrimination assay was used to genotype individual mites for the L925V mutation in the VGSC gene. For each population, phenotypic mortality data were compared with genotype frequencies to evaluate the degree of concordance between phenotypic and molecular indicators of resistance. Additional parameters were also evaluated, including the distribution of the resistance within an apiary and its evolution in French apiaries over several years. The phenotypic and genotypic assessments showed a strong concordance, with a correlation coefficient of 0.89 between taufluvalinate mortality rates and the frequency of the VGSC L925V mutation. Phenotypically resistant mites had a 63% probability of carrying the L925V mutation, indicating moderate predictive power for detecting resistance. In contrast, phenotypically susceptible mites exhibited a 97% probability of not carrying the mutation, confirming its high reliability for identifying susceptibility. The study also suggested that sampling approximately 50% of beehives within an apiary could provide a representative estimate of the resistance distribution. Overall, the study demonstrated that the L925V mutation accounts for most taufluvalinate resistance observed in the tested populations and represents a valuable molecular marker for susceptibility screening. All of these findings highlight the usefulness of integrating phenotypic and genotypic approaches to improve monitoring strategies and support more effective resistance management in beekeeping practices.