Summary Carthamus lanatus is a widespread and troublesome weed of crops and pastures in Australia. It is viewed as a target for both inundative and classical biological control. As fungal biological control agents are often specific in terms of their host range, the genetic variation within the host may be important in their successful deployment. The detection of genetic variation in populations of C. lanatus from New South Wales was studied using polymerase chain reaction (PCR)-based, intersimple sequence repeats (ISSR) analysis. DNA extracted from plant samples from 11 locations in New South Wales was compared with one sample from Western Australia and one accession of the closely related crop plant, safflower. The analysis indicated that there were two distinct groups of C. lanatus that correlated with location (northern and southern regions). Within-group genetic diversity, as determined by the Shannon genetic diversity index, was low at 0.33 in the southern group and 0.22 in the northern group. The presence of these groups may have implications for the biological control of C. lanatus using exotic fungi. In any screening of candidate agents, representatives of these populations should be included.