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Alligator weed, Alternanthera philoxeroides (Mart.) Griseb., is one of the worst invasive weeds in the world. In the present study, the genome size of seven forms/biotypes of alligator weed from Argentina (native area), the USA (introduced area), and China (introduced area) was analyzed using flow cytometers; the ribosomal DNA internal transcribed spacer region (ITS) was cloned and sequenced. We found that the genome size of A. philoxeroides ranged from 5.72 to 8.50 pg/2C nucleus, consisting of four levels of genome and three kinds of ploidy. Among them, three levels and two kinds (tetraploid and hexaploid) were found in samples from Argentina, two levels and one kind (pentaploid) in US samples, and one level and one kind (hexaploid) in samples from China. Contrary to the significant variation in genome size, we found that the intraspecific differentiation of the ITS sequences in the seven forms/biotypes was extremely low, and that six of them shared ITS sequences with no or one single substitution. These results show that the change of genome size played a major role in the differentiation of A. philoxeroides, and that forms/biotypes with higher ploidy seem to have stronger invasive ability. Furthermore, the differences of A. philoxeroides in ploidy between samples from the USA and China showed that alligator weeds from these two invaded areas certainly have different native progenitors.