The infusion of new genetic diversity from related species into domesticated types of cotton (Gossypium hirsutum L.) will greatly increase opportunities for genetic improvement. We report here the development of the aneuploid F1 chromosome substitution stocks in G. hirsutum for whole chromosomes and chromosome arms of G. mustelinum Miers ex Watt. These hypoaneuploid interspecific chromosome substitution stocks are an additional genetic resource for localization of genomic sequences, marker development, definition of linkage groups, and validation of genome maps. Hypoaneuploid plants that lack specific chromosomes or arms were detected by analysis of phenotypic syndromes and conventional meiotic metaphase-I configuration analysis of acetocarmine-stained microsporocytes ("pollen mother cells"), as well as by deletion analysis with chromosome specific SSR markers. Here, we report the development of 25 such hypoaneuploid hybrids, including 13 monosomic hybrids, each missing a different G. hirsutum chromosome (chromosome 1, 2, 4, 6, 7, 9, 10, 12, 16, 17, 18, 20, and 25, respectively), and 12 monotelodisomic (acrocentric) hybrids (Te05Lo, Te08Lo, Te11Lo, Te11sh, Te12Lo, Te14Lo, Te15Lo, Te20Lo, Te20sh, Te22Lo, Te22sh, and Te26sh) that are deficient for the respective distal segment of opposing G. hirsutum arms 5sh, 8sh, 11sh, 11Lo, 12sh, 14sh, 15sh, 20sh, 20Lo, 22sh, 22Lo, and 26Lo. Each of the interspecific F1s reported here is a major step toward a development of the respective backcross disomic substitution line. Such lines are individually and collectively powerful resources for targeted germplasm introgression, genetic dissection, and genetic improvement of complex traits.