Cotton (Gossypium spp.) is the most important textile fiber crop in the United States (US). Hybrid cotton is grown in several countries but the use of hybrids in the US has been limited due to seed production costs. The objective of this study was to investigate a novel method for the production of F2 cotton hybrids using honey bees as pollinators and the Roundup Ready® gene to facilitate identification of hybrid seed. This research was conducted from 2005 to 2007 in Louisiana. Six hybrid populations were developed between non-transgenic and transgenic lines manually or by caging with honey bees (Apis mellifera L.) as pollinators. In 2007, F1, F2, and parents were field tested in a randomized complete block design at two locations. All F1 hybrid populations exhibited heterosis compared to the best parent. The crosses LA1110023/PHY410R and ARKRM24-12-04/PHY410R exhibited the highest degree of high-parent heterosis for yield averaging 33.1% and 20.6% increases in the F1, respectively, and 20.9% and 19.5% increases in the F2, respectively. Fiber quality measurements did not display significant heterosis in the F2 population relative to the best parent. Using male contributors containing the Roundup Ready® trait for selection, conventional female lines, and honey bees as pollinators proved to be a viable method for developing F2 hybrid cotton lines.