Transgenic Bt cotton with insect resistance was introduced for commercial cultivation in India in 2002 (Barwale et al. 2004). Bt cotton expressing Cry1Ac is mainly toxic to the bollworm complex (Helicoverpa armigera, Pectinophora gossypiella, Earias insulana and E. vittella). The rapid adoption of Bt cotton technology in India imposes a strong selection pressure on the target pest with a risk of resistance development to Cry toxins including Cry2Ab following the introduction of dual gene BG-II cotton in 2006. Spotted bollworm, Earias insulana infests the cotton crop during the early season as a shoot borer and damages fruiting bodies during mid-season crop growth. Efficacy of Bt cotton hybrids BG-II (Cry1Ac + Cry2Ab) and Bt (Cry1Ac) through leaf (90–95-day-old crop) and square (80–85-day-old crop) bioassay under laboratory conditions, recorded 97–100 % mortality on leaves of BG-II, 93 % on Bt and 1.3–5.4 % on non Bt against 1-day-old larvae of Earias insulana. Observed mortalities on plant squares were 94–100 % in BG-II, 91 % in Bt and 1.3–5.6 % in non-Bt genotypes. Frequency of resistance alleles in field populations of Earias insulana to Cry1Ac protein by using F₂ screening procedure was also undertaken. In the present study on F₂ screening on 266 isofemale lines of Earias insulana collected from Sirsa (Haryana), Sriganganagar (Rajasthan) and Mansa (Punjab) during 2013–14, 2014–15 and 2015–16, no alleles could be detected conferring major resistance to Cry1Ac in the population of Earias insulana from all three locations. However, evidence for partial resistance alleles in F₂ generation of two isofemale lines from Sirsa (Haryana) population was recorded during 2013 but could not be reconfirmed.