The Varroa destructor (hereafter referred to as Varroa) is a major pest of honeybees that is generally controlled using pyrethroid-based acaricides. However, resistance to these insecticides has become a growing problem, driven by the acquisition of knockdown resistance (kdr) mutations in the mite's voltage-gated sodium channel (vgsc) gene. Resistance mutations in the vgsc gene, such as the L925V mutation, can confer resistance to pyrethroids like flumethrin and tau-fluvalinate. Monitoring genotypic resistance through molecular mutation screening is crucial for tracking and mitigating resistance spread. In this study, the frequency of resistance mutations in the vgsc was examined using a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) approach in Varroa populations sampled throughout the Mediterranean, Aegean, and Black Sea regions of Türkiye. Considering all the samples analyzed, the results demonstrated a mean resistance allele frequency of 83.29%, indicating a relatively high frequency of resistant alleles. We observed 94.58%, 85.71%, and 69.58% resistant allele frequencies in populations sampled from the Mediterranean, Aegean, and Black Sea regions, respectively, in our study. The results of our investigation demonstrated substantial regional variations in the frequencies of resistant alleles among Varroa populations throughout Türkiye, with notably elevated resistance levels observed in the Mediterranean and Aegean regions. Due to the significant resistance mutation frequency differences between both provinces and regions, long-term monitoring of resistance alleles and the planning of regional control strategies are required for effective control of this pest.