The flax rust resistance gene L, a nucleotide binding site, leucine-rich repeat (NBS-LRR) class of plant resistance gene, has 12 characterized alleles with different gene-for-gene resistance specificities. Here the specificities of presumptive L1, L5, L8 and L11 genomic clones are confirmed by transgenic expression. L6 and L11 differ by 33 amino acids, 32 in the LRR region and one in the C-terminal non-LRR region, and recognize unrelated avirulence proteins, AvrL567 and AvrL11, respectively. To analyse the specificity differences, 13 L6L11 recombinant genes were constructed in vitro and tested in transgenic flax for resistance to F2 progeny of rust strain CH5, in which the unlinked avirulence genes AvrL567 and AvrL11 segregate. The data show that the single C-terminal non-LRR region polymorphism is not involved in L6–L11 specificity differences, that polymorphisms necessary for specificity are spread throughout the LRR region and that some polymorphisms essential for L11 are not essential for L6. Seven ‘null’ recombinants expressed no resistance when tested with CH5-derived rusts. These were tested for new resistance specificities by inoculation with a strain of rust, Bs-1, which is distantly related to CH5 and which potentially carries a different range of avirulence specificities. The ‘null’ recombinant L6L11RV, which differs from L6 and L11 by its susceptibility to CH5, was resistant to strain Bs-1. The specificity difference is due to a reduction in the number of AvrL567 variants recognized by L6L11RV compared with L6 and not due to recognition of an unrelated Avr gene product in strain Bs-1.