The tobacco aphid, Myzus nicotianae (Blackman), is a key pest of tobacco, reducing the yield and quality of severely infested crops. The organophosphate (acephate) and the carbamates (aldicarb / methomyl) are alternative insecticides registered and used throughout Virginia, and adjacent states, as a resistant management strategy for imidacloprid-tolerant tobacco aphid populations. Recent evidence suggests that tobacco aphid populations are becoming less susceptible to both organophosphate and carbamate insecticides. However, little information exists regarding the mechanism(s) conferring this decreased insecticide susceptibility or any changes that may have occurred for the past 15 years. In this study, colonies of M. nicotianae were collected from 18 Virginia counties, and nine other states, between 2004 and 2007. The susceptibility of tobacco aphids to both acephate and methomyl was evaluated using a leaf dip bioassay. Biochemical analyses were performed on the aphid colonies to determine activities of enzymes, including cytochrome P450 monooxygenases, glutathione S-transferases, and general esterases between susceptible and resistant colonies. The molecular characterization of those genes that may contribute to the loss of insecticide susceptibility will be discussed. Elucidation of biochemical mechanisms conferring resistance and, therefore, a better understanding of the resistance factors is necessary for managing insecticide resistance in the tobacco aphid.