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Amitraz, a formamidine acaricide, plays an important role in the control of the southern cattle tick, Boophilus microplus (Canestrini), and other tick species that infest cattle, dogs, and wild animals. Although resistance to amitraz in B. microplus was previously reported in several countries, the actual measurement of the level of amitraz resistance in ticks has been difficult to determine due to the lack of a proper bioassay technique. We conducted a survey, by using a newly reported technique that was a modification of the standard Food and Agriculture Organization larval packet test, to measure the levels of resistance to amitraz in 15 strains of B. microplus from four major cattle-producing states in Mexico. Low-order resistance (1.68- to 4.58-fold) was detected in 11 of those strains. Our laboratory selection using amitraz on larvae of the Santa Luiza strain, which originated from Brazil, achieved a resistance ratio of 153.93 at F6, indicating the potential for high resistance to this acaricide in B. microplus. Both triphenylphosphate and piperonyl butoxide significantly synergized amitraz toxicity in both resistant and susceptible tick strains. Diethyl maleate synergized amitraz toxicity in one resistant strain but had no effect on the susceptible strain and had minor antagonistic effects on two other resistant strains. Target site insensitivity, instead of metabolic detoxification mechanisms, might be responsible for amitraz resistance observed in the Santa Luiza strain and possibly in other amitraz resistant B. microplus ticks from Mexico. The Santa Luiza strain also demonstrated high resistance to pyrethroids and moderate resistance to organophosphates. Multiple resistance shown in this strain and other B. microplus strains from Mexico poses a significant challenge to the management of B. microplus resistance to acaricides in Mexico.