Technical Abstract: This study was performed to obtain an emerging view of the epidemiology of acaricide resistance in populations of R. microplus from Mato Grosso do Sul state in Brazil. Twenty four tick samples were collected from cities in the state where farmers reported concerns about resistance or failure of tick control management. Those ticks were subjected to an in vitro resistance detection assay using the adult immersion test (AIT). The efficacies of alpha-cypermethrin, cypermethrin, and amitraz treatments were generally poor on samples collected throughout the state. Efficacy =90% in the AIT was obtained with DDVP + Chlorpyriphos (20 of 21 samples), Dichlorvos + Cypermethrin + Piperonyl Butoxide (10 of 16 samples), and Cypermetrin + Citronella + Chlorpyriphos + Piperonyl Butoxide (20 of 21 samples). A PCR assay was used, which detects the presence of a target site pyrethroid resistance-associated sodium channel gene mutation, to evaluate larvae from three populations that were diagnosed as pyrethroid resistant by AIT. The PCR assays showed the pyrethroid resistance-associated gene mutation was absent from these three populations. Thus, either metabolic resistance mechanisms are present in these ticks or a unique sodium channel gene mutation is responsible for pyrethroid resistance. Our study confirms that the emergence of resistance is a constant challenge to the cattle industry and the development of resistance continues to be a major driver for the need to discover and develop tickicides with new modes of action.