Tobacco mosaic virus (TMV)-resistant flue-cured tobacco (Nicotiana tabacum L.) cultivars have been developed using the N gene derived from N. glutinosa L. Their adoption has been low, however, because of unfavorable linkage drag effects. Strategies to overcome this problem might include pursuit of alternative introgression events and/or use of molecular markers for selection against deleterious alien chromatin. Previous workers demonstrated the presence of a TMV-resistance mechanism on more than one chromosome of the tobacco genome. The objectives of this research were to determine the relative genomic positions of TMV resistance loci in a set of 12 TMV-resistant tobacco accessions and to use amplified fragment length polymorphism (AFLP) markers for characterization of this material with respect to linked alien chromatin. Five accessions were found to carry a TMV resistance gene on chromosome H. Seven accessions were found to carry a resistance factor on an alternative chromosome. Polymerase chain reaction results indicated that the N gene from N. glutinosa is responsible for resistance in all 12 accessions. A set of 168 AFLP markers specific to the N. glutinosa donor chromosome was identified and used to reveal variability among the 12 accessions for the relative amounts of N. glutinosa chromatin linked to the N gene. The relative propensity for crossing over within the alien segment when in different genomic positions was evaluated in BC 1F1 families derived from three different accessions. Lines possessing the N gene on chromosome H may be of greater practical value because of relatively smaller introgressed alien segments and increased potential for obtaining crossover events within the segments. © Crop Science Society of America.