Background The perennial cotton species Gossypium hirsutum L. r. marie-galante Hutch. and Gossypium barbadense L. are sources of variability for creating modern cotton varieties. However, these species are similar in shape and easily confused in young stages of their life, leading to di culties in identi cation based on their morphology. Thus, in this study, an Inter-Simple Sequence Repeat (ISSR) marker system was used as a measure of genetic differentiation among 28 genotypes of perennial cotton from a Brazilian collection. Methods and Results A set of eleven ISSR primers yielded 101 bands, of which 48 (47.5%) were polymorphic. The mean values of polymorphism information content (PIC=0.304) and polymorphism (P=45.8%) showed that the ISSR primers are moderately informative. The ISSR markers exposed the high genetic differentiation (G ST = 0.598) and a low level of gene ow (N m = 0.338) between species, which suggests a pattern of reproductive isolation. The Cluster Analysis, based on Nei's genetic identity, clustered the 28 genotypes into two groups consistent with the taxonomical delimitation, occurrence mode (semi-domesticated and improved types) and partial concordance with geographic origin. Bayesian model-based structural analysis also suggests the existence of two genetic groups (Delta K = 2) and high membership likelihood (Q>0.98).Conclusions The ISSR marker system offers a new molecular approach to differentiate the G. hirsutum L. r. marie-galante and G. barbadense L. species. This study can expand the molecular marker resources for the identi cation and improvement of our knowledge about the genetic relationships between perennial cotton genotypes from Brazilian collections.G. hirsutum L. r. marie-galante Landrace Paraíba, Brazil G. hirsutum L. r. marie-galante Landrace