Downy mildew (DM) caused by Peronospora arborescens, is a serious disease in opium poppy (Papaver somniferum), which has a world-wide spread. The establishment of DM-resistant cultivars appears to be a sustainable way to control the In this paper, we present the results of a study aimed at the identification of amplified fragment length polymorphism (AFLP) markers for DM-resistance in opium poppy. Three opium poppy genotypes (inbred over about 10 years): Pps-1 (DM-resistant), Jawahar-16 (DM-susceptible) and H-9 (DM-susceptible) were crossed in a diallel manner and the F(1) progeny along with the parents were subjected to AFLP analysis of chloroplast (cp) and nuclear DNA with seven and nine EcoRI / MseI primer combinations, respectively. cpDNA AFLP analysis identified 24 Pps-1 (DM-resistant)-specific unique fragments that were found to be maternally inherited in both the crosses, Pps-1 x Jawahar-16 and Pps-1 x H-9. In the case of nuclear DNA AFLP analysis, it was found that 17 fragments inherited from Pps-1 were common to the reciprocal crosses of both (i) Pps-1 and Jawahar-16 as well as (ii) Pps-1 and H-9. This is the first molecular investigation on the identification of polymorphism between DM-resistant and DM-susceptible opium poppy genotypes and development of DM-resistant opium poppy genotypespecific AFLP markers. These AFLP markers could be used in future genetic studies for analysis of linkage to the downy mildew resistance trait.