The close relationship between Gossypium species (Malvales) and Arabidopsis (Brassicales) determined by molecular phylogeny might facilitate the identification of regulatory mechanisms for trichome development in cotton leaves. Focus of this study is on the evaluation and quantification of differences in trichomes on cotton leaves with varying degrees of leaf pubescence. Trichomes were evaluated using scanning electron and light microscopy on both fixed and live samples. Trichome quantity and complexity were measured on mainstem leaves and usually found to increase on new leaves as they develop progressively up the plant. Glandular trichomes (GTs) were found on all mainstem leaves examined and were not affected by various loci that either increased or inhibited the expression of non-GTs (NGTs or hairs). Neither GTs nor NGTs on Pilose leaves were affected by the expression of N1N1 genotype, which removes approximately 75% of all lint and 100% of all fuzz on cotton ovules. The abaxial side of the leaf usually had more GTs and NGTs. These data indicate that at least two regulatory networks exist in cotton leaves for trichome initiation, one for GTs and the other for NGTs. A third regulatory network is likely for the initiation of ovule trichomes; however, this third network might share genetic components with the NGTs. Future studies will use four very pubescent cotton lines identified in this survey for further genetic evaluations by crossing with three fiberless ovule lines.