Due to the lack of molecular marker showing polymorphism inside the Nicotiana tabacum germplasm commonly used by tobacco breeders, no reliable molecular method was available to identify tobacco lines. Use of the AFLP method is changing this situation. Automated data entry and analysis are needed to realize the full potential of AFLP technology. To this end, the capillary electrophoresis Applied Biosystems ABI 310 was selected. Among 3 tobacco lines, it appeared that 11 primer pairs were sufficient to identify 48 polymorphic markers. Use of these markers within a set of 131 tobacco varieties from various types (flue-cured, dark air-cured, oriental, burley) led to a phylogenetic tree, highlighting different groups sharing a common genetic background. Combining marker data and disease resistance data in a multidimensional statistical analysis revealed that several AFLP markers could be connected to resistance genes (resistance to black root rot caused by Thielaviopsis basicola , "va" deletion conferring resistance to PVY virus, and resistance to blue mold caused by Peronospora tabacina ). The use of additional restriction enzymes should be in the future an opportunity to obtain a sufficient number of markers in order to saturate a genetic map and to characterize quantitative traits related to tobacco quality.