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P>Evolutionary transitions from outcrossing to selfing occur commonly in heterostylous genera. The morphological polymorphisms that characterize heterostyly provide opportunities for different pathways for selfing to evolve. Here, we investigate the origins and pathways by which selfing has evolved in tristylous Eichhornia paniculata by providing new evidence based on morphology, DNA sequences and genetic analysis. The primary pathway from outcrossing to selfing involves the stochastic loss of the short-styled morph (S-morph) from trimorphic populations, followed by the spread of selfing variants of the mid-styled morph (M-morph). However, the discovery of selfing variants of the long-styled morph (L-morph) in Central America indicates a secondary pathway and distinct origin for selfing. Comparisons of multi-locus nucleotide sequences from 27 populations sampled from throughout the geographical range suggest multiple transitions to selfing. Genetic analysis of selfing variants of the L- and M-morphs demonstrates recessive control of the loss of herkogamy, although the number of factors appears to differ between the forms. Early stages in the establishment of selfing involve developmental instability in the formation of flowers capable of autonomous self-pollination. The relatively simple genetic control of herkogamy reduction and frequent colonizing episodes may often create demographic conditions favouring transitions to selfing in E. paniculata. New Phytologist (2009) 183: 546-556doi: 10.1111/j.1469-8137.2009.02937.x.