The present study, comprising an evaluation of cotton amphidiploid hybrids and their families for yield and fiber quality traits, commenced from 2007 to 2022 at the Chirchik State Pedagogical University, Tashkent, Uzbekistan. The amphidiploid hybrids’ creation was through interspecific hybridization of Gossypium hirsutum subsp. euhirsutum cultivar Kelajak × F1 (G. arboreum subsp. perenne × G. arboreum subsp. obtusifolium var. indicum) and experimental polyploidy, with genetically enriched new genotypes obtained. According to genetic variability, a considerable variation showed based on F1–F6 populations belonging to various clusters for the traits boll weight (2.3–6.21 g), 1000-seed weight (67.3–125.529 g), fiber length (24.9–34.4125 mm), fiber yield (31.4%–40.26%), and fiber index (6.3–7.5875 g). Furthermore, among the F6 G. hirsutum subsp. euhirsutum cultivar Kelajak × F1 (G. arboreum subsp. perenne × G. arboreum subsp. obtusifolium var. indicum) combination families, the promising genotypes selected gave the highest trait values for boll weight (6.6 ± 0.13 g – Family- 41), 1000-seed weight (125.8 ± 3.48 g – Family-59), fiber yield (40.3% ± 0.65% – Family-59), fiber index (8.5 ± 0.23 g – Family-59), and fiber length (34.5 ± 0.16 mm – Family-8). Genotype evaluation using cluster analysis allows the prediction of cotton families with a better combination of traits. Introgressive hybrids created based on the early maturing families, viz., Family-5, Family-8, Family- 13, Family-14, Family-41, and Family-59, with high yields and fiber indices attained inclusion in the Cotton Gene Pool, Uzbekistan (Certificate of the Academy of Sciences of the Republic of Uzbekistan No. 4/1255-2635 dated November 26, 2020). The promising families selected based on their best performance included F5 (G. hirsutum L. × F1 [G. arboreum L. × G. arboreum L.], Family-5, Family-8, Family-13, Family-14, and Family-59) and F6 (G. hirsutum L. × F1 [G. arboreum L. × G. arboreum L.], Family-5, Family-13, and Family-59), with the said cotton gene pool as valuable recombinants benefiting future breeding programs.