The Varroa mite, Varroa destructor is an ectoparasite that parasitizes honey bees. The widespread usage of acaricides, particularly fluvalinate, has resulted in the emergence of resistance in Varroa mite populations all over the world. The goal of this study is to track fluvalinate resistance in Varroa mite field populations in Korea using both bioassay and molecular markers. To accomplish this, a residual contact vial (RCV) bioassay for on-site resistance monitoring was developed. Early mortality evaluation in the RCV bioassay was effective for reliably separating mites with the knockdown resistance (kdr) genotype, but late mortality evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 revealed potential resistance development in four populations. Quantitative sequencing was used as an alternate method to examine the frequency of the L925I/M mutation in the voltage-gated sodium channel (vgsc), which is related with the fluvalinate kdr phenotype. While the mutation was not present in Varroa mite populations in 2020, it appeared in 2021, rose in frequency in 2022, and was practically ubiquitous across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation emphasizes the critical necessity to replace fluvalinate with alternate acaricides, such as fenpyroximate, coumaphos, and amitraz. A few novel vgsc mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.