The possibility of inducing embryo development after pollination of F₁ interspecific cotton hybrids (Gossypium barbadense × Gossypium hirsutum) and their reciprocals with pollen from Hibiscus cannabinus was investigated. For this, flowers of F₁ plants from 4 G. barbadense × G. hirsutum interspecific hybrids (B403 × Acala Sindos, Carnak × 4S, B403 × Coker 310, and Carnak × Acala Sindos) and their reciprocals grown in the field were pollinated with pollen from Hibiscus cannabinus. From the 443 pollinated flowers, 276 were left on the plant to grow naturally, and 167 were collected 5 days after pollination. Young ovules from the collected buds were cultured in vitro for embryo development. It was observed that, from the buds left to grow naturally on the mother plant, 21 bolls reached maturity. The mature bolls originated only from the 4 G. barbadense × G. hirsutum hybrids and contained 82 mature seeds. Finally, 38 plants (Pa0) were produced. From the in-ovule culture method, 10 young embryos were isolated from both G. barbadense × G. hirsutum and G. hirsutum × G. barbadense hybrids and finally 3 plants were produced. The plants produced from both approaches originated only from the G. barbadense × G. hirsutum hybrids. These plants exhibited morphological traits from both cotton species and they were partially fertile. No signs of H. cannabinus morphological traits were observed in the plants produced. Root-tip chromosome counts revealed that chromosome number among cells of the Pa0 plants ranged from 27 to 42 and the difference in chromosome number observed among cells of the same plant ranged from 1 to 3. The chromosome number, however, was increased progressively from generation to generation and in Pa3 it ranged from 46 to 52. Plants with 52 chromosomes were identified even from the Pa1 generation. In addition, flow cytometric analysis indicated that the parental plants had a similar DNA profile to the F₁ and F₂ interspecific hybrids but a different one from the Pa0 plants. Thus, alien pollination of cotton flowers from interspecific (G. barbadense × G. hirsutum and reciprocals) hybrids with pollen from H. cannabinus most likely induced parthenogenetic (Pa) egg cell development which, after a progressive chromosome increase, produced fully fertile plants with most of the cells at the tetraploid or near-tetraploid level. It was concluded that a combination of the in situ boll development with an optimised in vitro ovule culture technique could establish the ‘cannabinus method’ in cotton, as a method for the production of genotype-independent partial interspecific lines.