Gfi-1 is a C(2)H(2)-type zinc finger protein that is a transcriptional repressor in vertebrates and has been implicated in control of CYP6D1 expression in house flies (Musca domestica). A 15 bp insert, which disrupts a putative mdGfi-1 binding site in the CYP6D1v1 promoter has been implicated as a cause of increased expression of CYP6D1, and thus insecticide resistance. Using electrophoretic mobility shift assays we demonstrate that the CYP6D1 promoter from susceptible strains binds mdGfi-1. The 15 bp insert that interrupts the mdGfi-1-binding site in insecticide-resistant strains reduces the amount of mdGfi-1 binding by 9- to 20-fold, consistent with the role of mdGfi-1 in resistance. Partial sequences of mdGfi-1 (spanning the first intron) from individual houseflies from 11 different strains revealed the presence of 23 alleles. There was no consistent difference in the mdGfi-1 alleles between susceptible and CYP6D1-mediated insecticide-resistant strains, indicating that mdGfi-1 alleles were not likely involved in resistance. Polymorphisms were used to map mdGfi-1 to autosome 1. Quantitative real time PCR (qRT-PCR) revealed Gfi-1 expression was higher in the thorax compared to the head and abdomen, and varied between life stages and between strains. However, similar levels of mdGfi-1 were detected in susceptible and resistant adults suggesting that altered levels of mdGfi-1 were not likely a cause of insecticide resistance. The significance of these results to understanding insecticide resistance is discussed.