Parthenium hysterophorus is an invasive weed native to tropical and subtropical America. Since its introduction into China in 1926, this weed has been limited to southern areas (18°–25°N). However, new populations were recently reported in Shandong (35°N). The origin of this geographically isolated population remains unclear. In this study, we used inter simple sequence repeat and chloroplast DNA sequence markers (trnQ-5’rps16) to investigate the genetic structure of 18 invading populations of P. hysterophorus and to examine the genetic relationship of Shandong and southern populations. Our data show that Shandong population is genetically different and has apparently higher levels of genetic diversity than most populations in southern China. Both neighbour-joining clustering and principal coordination analysis clearly separated Shandong from other populations. Chloroplast DNA sequence analysis further confirmed that the Shandong population was characterised by a unique haplotype that was not found in southern populations. We conclude that the population in Shandong may have been independently introduced from areas outside China. As P. hysterophorus has a broad potential distribution in China, attention should be paid to both international and domestic quarantine to prevent new introductions and curtail the possible hybridisation and gene flow between populations in different regions.