A total of 85 wild plants were collected in different areas of southern and south‐eastern Brazil, and studied for nrDNA internal transcribed spacers (ITS). Thirty‐two of them were also investigated for two other nuclear (G3pdh and LEAFY), as well as eight plastid systems. Sixty‐one ITS sequence types were identified, in a region of 412 bp, whereas 42 G3pdh haplotypes were found in a region of 1010 bp, and 23 LEAFY haplotypes in a region of 565 bp. GC content was higher in ITS (65%) than in the two other nuclear regions (40–42%), whereas nucleotide diversity was markedly higher in G3pdh than in the other two. No variability was found in the plastid systems. Most of the nuclear molecular diversity (56–86%) occurs at the intrapopulation level, and there is clear evidence of population expansion and little geographical structure. Historical information and field studies indicate that Passiflora alata is actively colonizing previously unoccupied areas in southern Brazil. High indices of intrapopulation variability were found in the region that is being colonized, similar to those found in areas where P. alata is native. Multiple introductions, as well the species’ genetic structure and human interference, may explain these findings. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 611–630.