Diploid cotton Gossypium arboreum (2n = 2x = 26, AdAd) is a valuable genetic resource to improve widely cultivated American cotton G. hirsutum (2n = 4x = 52, AtAtDtDt). In this study, successful generation of four unique interspecific hybrids (G. hirsutum × G. arboreum) was confirmed through morphological, cytological, and molecular characterization. The morphological evaluation included different stem and leaf characters, trichome density, floral characters, and cotton leaf curl disease reaction. Interspecific hybrids were recorded with either complete dominance of some characters or an intermediate expression. However, variation among the F1s was observed for some traits, especially for pollen size and fertility. Two of the four F1s were found to possess relatively bigger pollen sizes with partial fertility which enabled their utilization as male parents in backcrosses. Mitotic analysis of already established F1s was made possible through the induction of fresh roots by air layering. Of the four hybrid plants, two were triploid (2n = 39) and the other two were tetraploids (2n = 52), the latter case probably was a result of the fusion of normal n (AtDt) gametes from female parent (G. hirsutum) and unreduced gametes 2n (AdAd) from male parent (G. arboreum). Meiotic irregularities in interspecific hybrids were also observed. Successful amplification of polymorphic parental bands in all four F1s using simple sequence repeat markers added another line of evidence supporting their hybridity. Results of this experiment provided useful insight and base material to introgress leaf-hopper tolerance from G. arboreum to G. hirsutum.