The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), is a major vector of tick fever organisms affecting cattle in many parts of the world, including Australia, Africa, and South America. Control of the southern cattle tick through acaricide use is an important approach in disease management, Resistance has emerged to many of the acaricides currently and previously used, including the cyclodienes. Although cyclodiene resistance mechanisms have been characterized in many insect species, this report is the first to identify mutations associated with dieldrin resistance in the cattle tick. A novel two base pair mutation in the GABA-gated chloride channel gene has been identified at position 868–9 and causes a codon change from threonine to leucine. Analysis of a small number of field-collected samples resistant to dieldrin shows this mutation has been maintained without selection pressure since the withdrawal of dieldrin in Australia >20 yr ago. The mutation is not found in other laboratory-maintained strains of R. microplus that were subject to selection pressure with various acaricides.