Amplified fragment length polymorphism (AFLP) markers were employed to detect genetic variation among species of Papaver (section Oxytona ) and assess genetic fidelity between in vitro cell lines of Papaver bracteatum and mature plants derived from the propagation of their callus cultures. Regenerated plants exhibited morphological and phytochemical characteristics dissimilar to those of their source material. Thebaine, the dominant alkaloid produced by Papaver bracteatum, was not detected in capsules from mature regenerated accessions, indicating that there may have been a loss of genetic uniformity. Instead, the dominant alkaloid produced by the regenerated plant was shown to be isothebaine (by TLC and GC/MS), a metabolic characteristic of P. pseudo-orientale. A Neighbor-Joining tree constructed from AFLP fingerprints distinctly separates the three species of Oxytona while firmly grouping the in vitro-cultured plants with P. pseudo-orientale. Additionally, phytochemical data and chromosome counts indicate that the seed used to initiate cultures was of hybrid origin and that the loss in genetic uniformity was not due to somaclonal variation occurring during the in vitro culture process. AFLP fingerprinting was therefore able to differentiate Oxytona species and investigate allopolyploidy in closely related Papaver species.