Rhipicephalus sanguineus s.l. (Latreille), is a vector of multiple disease-causing pathogens to humans and dogs. Permethrin and fipronil are two acaricides used to manage R. sanguineus s.l. infestations. Homeowners have reported treatment failures in managing brown dog ticks using permethrin and fipronil based products. Previous studies demonstrated that high permethrin resistance in some R. sanguineus s.l. populations was due to metabolic detoxification and target site insensitivity. In this study, three R. sanguineus s.l. strains, one from a laboratory colony (NC) and two colonies originally collected from Florida (FL) and California (CA), were evaluated for resistance expression against permethrin and fipronil. Metabolic detoxification mechanisms were evaluated in the FL strain using three synergists, while a polymerase chain reaction assay was used to detect a resistance mutation in all strains.The NC strain was susceptible to both permethrin and fipronil, while both the FL and CA strains exhibited high resistance to permethrin and tolerance to fipronil. The synergist tests and PCR results indicated that the FL strain utilized both metabolic resistance and target site insensitivity against permethrin, while the CA strain was documented to have the target-site insensitivity resistant allele. This study confirmed permethrin resistance in both California and Florida populations and its persistence in Florida populations, although its susceptibility can potentially be increased by adding a synergist. Fipronil resistance was not detected suggesting this acaricide may provide suitable tick control.