The Australian arid zone Gossypium species are desert dwellers that make up the G genome, an important part of the tertiary germplasm pool of the genus. The current distribution of G. australe extends from the coast of Western Australia to central Queensland, whereas G. nelsonii and G. bickii are primarily distributed in the central part of the Northern Territory extending into western Queensland. Field observations in their natural habitats indicate that natural hybridization can occur among these species when they found growing sympatrically. This is the initial report of a study to determine the molecular diversity among the species and to determine if molecular evidence supports the field observations of natural hybridization. Our hypothesis is that the construction of roads that are used by roadtrains and other high speed vehicles as allowed G. australe, which has wind distributed seeds, to be carried eastward and established in formerly isolated habitats of G. nelsonii and G. bickii. Greenhouse work indicates that there is no sexual barrier to hybridization, so when these species grow adjacent to one another, in the presence of pollinators, natural hybrids can be expected. The hypothesis has the implicit assumption that any natural hybridizations are recent in origin, hence plants should be near the media of the two parental species. Accessions representing the distribution range of each species were selected for examination as well as accessions noted as being from sites with possible hybrization (103 total). G. sturtianum collected from an area sympatric to the other species was included as an “outgroup.” Three plants of each accession were grown in a greenhouse and DNA isolated from young leaves of each plant. The DNA was fingerprinted with the techniques use to detect Amplified Fragment Length Polymorphisms (AFLP) (38 fragments from two primer combinations). A dendrogram was constructed with PAUP* v. 4.2b software using Parsimony. The dendrogram showed that each of the three species formed a distinct cluster, however, two groups of accessions grouped outside of these. One clustered between G. australe and G. nelsonii and the other between G. australe and G. bickii. Two accessions each that fall within this group were collected as possible hybrids. Thus, this dendrogram, based on limited data, strongly suggests that the other accessions in each cluster are also the result of interspecific hybridizations and introgressions. With additional markers we hope to identify the origin of the contributing G. australe parents in order to refine and verify the hypothesis given above. G. australe is the most widely distributed of the Australian arid zone Gossypium species while, G. nelsonii has the most restricted range and the least diversity of the three species. Because G. australe appears to have recently invaded the habits of G. nelsonii and G. bickii through the activities of man, in the medium term, it is possible that the distinctiveness and genetic diversity of these latter two species may be lost through introgression of G. australe genes.