The major cluster of disease resistance genes in lettuce (Lactuca sativa) contains at least nine downy mildew resistance genes (Dm) spanning a genetic distance of 20cM and a physical distance of at least 6 Mb. Nine molecular markers that were genetically tightly linked to Dm3 were used to analyze nine independent deletion mutants and construct a map of the region surrounding Dm3. This analysis identified a linear order of deletion breakpoints and markers along the chromosome. There was no evidence for chromosomal rearrangements associated with the deletions. The region is not highly recombinagenic and the deletion breakpoints provided greater genetic resolution than meiotic recombinants. The region contains a mixture of high-and low-copy number sequences; no single-copy sequences were detected. Three markers hybridized to low-copy-number families of sequences that are duplicated predominantly close to Dm3. This was not true for sequences related to the triose-phosphate isomerase gene; these had been shown previously to be linked to Dm3, as well as to two independent clusters of Dm genes, and elsewhere in the genome. Two spontaneous mutants of Dm3 were identified; several markers flanking Dm3 are absent in one of these two mutants. The stability of the Dm3 region was also studied by analyzing the genotypes of diverse related cultivars. The 1.5 Mb region surrounding Dm3 has remained stable through many generations of breeding with and without selection for Dm3 activity.