The tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is one of the most important pests of cotton, Gossypium hirsutum L, that has become resistant to a wide range of synthetic insecticides. CrylAc-expressing cotton has proven its effectiveness against this insect since its introduction in North America in 1996. However, the constant exposure of tobacco budworm to this protein toxin may result in the development of resistance to it. To estimate the frequency of alleles that confer resistance to a 1.0 microg of Bacillus thuringiensis Cry1Ac diagnostic concentration in field-collected insects, the second generation (F2) of 1,001 single-pair families from seven geographical regions representing 2,202 alleles from natural populations was screened in 2006 and 2007 without finding major resistant alleles. Neonates of 56 single-pair families were able to develop to second instar on the diagnostic concentration in the initial screen, but only seven of these lines did so again in a second confirmatory screen. Minor resistance alleles to Cry1Ac may be quite common in natural populations of H. virescens. Our estimated resistance allele frequencies (0.0036-0.0263) were not significantly different from a previously published estimate from 1993. There is no evidence that H. virescens populations have become more resistant to Cry1Ac.