Understanding the breeding system and population genetic structure of invasive weed species is important for biocontrol, and contributes to our understanding of the evolutionary processes associated with invasions. Hieracium lepidulum is an invasive weed in New Zealand, colonising a diverse range of habitats including native Nothofagus forest, pine plantations, scrubland and tussock grassland. It is competing with native subalpine and alpine grassland and herbfield vegetation. H. lepidulum is a triploid, diplosporous apomict, so theoretically all seed is clonal, and there is limited potential for the creation of variation through recombination. We used intersimple sequence repeats (ISSRs) to determine the population genetic structure of New Zealand populations of H. lepidulum. ISSR analysis of five populations from two regions in the South Island demonstrated high intrapopulation genotypic diversity, and high interpopulation genetic structuring; PhiST = 0.54 over all five populations. No private alleles were found in any of the five populations, and allelic differentiation was correlated to geographic distance. Cladistic compatibility analysis indicated that both recombination and mutation were important in the creation of genotypic diversity. Our data will contribute to any biocontrol program developed for H. lepidulum. It will also be a baseline data set for future comparisons of genetic structure during the course of H. lepidulum invasions.