A three-species hybrid named HTL including Gossypium hirsutum L. [2n = 4 x = 52, (AD), genome] was created using the pseudophyletic introgression method with G. longicalyx Hutch. & Lee (2n = 2x = 26, F, genome) as donor parent and G. thurberi Tod. (2n = 2x = 26, D1 genome) as bridge species. The new hybrid was totally self-sterile and its interspecific status was confirmed using simple sequence repeat markers and cytogenetic analysis. Cytogenetic studies showed that its chromosome configuration was 2n = 52 = 14.13 I + 15.10 II + 1.03 III + 0.9 IV + 0.03 V + 0.13 VI (where I, II, III, IV, V and VI are univalents, bivalents, trivalents, tetravalents, pentavalents and hexavalents, respectively). Prospects for successfully exploiting the HTL hybrid in breeding programmes are discussed.