Horn flies, Haematobia irritans, a major cattle pest in the USA, cause substantial economic losses and current control methods rely heavily on insecticides. Three horn fly populations were evaluated for insecticide susceptibility to permethrin, &beta / -cyfluthrin, and diazinon. Susceptibility was variable by population, with the greatest resistance exhibited by a 66-fold resistance ratio (RR) to permethrin and > / 14-fold RR to diazinon. Mechanisms of resistance were determined using molecular techniques and enzymatic assays. The knockdown resistance (kdr) genotype (L150F) associated with pyrethroid resistance, and a G262A mutation in acetylcholinesterase, previously associated with organophosphate resistance, were found in all field populations evaluated. Insensitivity of diazoxon at the acetylcholinesterase (AChE) target site was significantly different in horn flies from one of the field sites. For metabolic detoxifying enzymes, cytochrome P450 nor general esterases showed a significant difference between field strains and a laboratory susceptible strain. Pyrethroid resistance was likely due to the presence of the L150F mutation in the population. In vitro studies targeting the AChE enzyme did not support the notion that the G262A mutation was the sole cause of resistance to organophosphates, and, therefore, the exact resistance mechanism to diazinon was not able to be confirmed.