[en] Gossypium australe F. Muell (2n + 2x = 26) is a wild perennial species possessing agronomic useful traits that would be interesting to introgress into G. hirsutum L. (2n = 4x = 52), the main cultivated cotton species. To isolate monosomic alien addition lines (MAALs) of G. australe in G. hirsutum, the [2(G. hirsutum X G. australe) X G. hirsutum] pentaploid (2n = 5x = 65) was backcrossed as male parent to G. hirsutum. Analysis of 42 BC1 plants and seven alien addition lines, already available, with 150 SSR markers developed from G. hirsutum revealed a cross-species amplification rate of 100% and a polymorphism rate of 56%. Eighty polymorphic SSR markers generated 87 G. australe-specific loci that have been assigned by a hierarchical cluster analysis to 13 linkage groups corresponding to the 13 chromosomes of G. australe. Analysis by SSR markers and genomic in situ hybridization of the self-progeny of disomic alien addition lines, backcross progeny of the pentaploid, allowed the isolation of five new MAALs.