Genes at the M locus in flax (Linum usitatissimum) that confer resistance to flax rust (Melampsora lini) occur in complex haplotypes containing up to 15 related genes or gene fragments. We have cloned two additional functional resistance genes at this locus, M1 and M3, by transposon tagging and candidate gene approaches, and investigated the genetic relationships between four genes (M, M1, M3 and M4) by recombination analysis. M1 and M3, like M, are members of the nucleotide binding site, leucine-rich repeat (NBS-LRR) family. Comparisons of the predicted M1 and M3 amino acid sequences with M and L6 reveal that: (i) M1 contains four additional LRRs, probably as a result of an unequal crossover event between duplicated regions; (ii) M1 shares large segments of exact identity with M and M3, indicative of intragenic recombination events; and (iii) a large number of amino acid differences are scattered throughout the M, M1 and M3 proteins. Recombination analysis (here and in previous studies) has revealed that M readily recombines with M1, M3 and M4, whereas these three genes fail to recombine despite large family sizes (>5800) in two test-cross families, suggesting that they may occupy allelic positions in the gene cluster. Several restriction fragment length polymorphism markers within or near the M locus were mapped with respect to seven crossover events between M and M1. The results of this and previous studies provide evidence of structural differences between: (i) homoeologous loci in the different genomes of flax; (ii) different haplotypes at the M locus; (iii) different resistance genes in the M group; and (iv) the flanking regions downstream of M locus resistance genes.