Most natural Arabidopsis thaliana accessions are susceptible to one or more isolates of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). However, Arabidopsis C24 has proved resistant to all Hpa isolates tested so far. Here we describe the complex genetic basis of broad-spectrum resistance in C24. The genetics of C24 resistance to three Hpa isolates was analyzed by segregation analysis and quantitative trait locus (QTL) mapping on recombinant inbred and introgression lines. Resistance of C24 to downy mildew was found to be a multigenic trait with complex inheritance. Many identified resistance loci were isolate-specific and located on different chromosomes. Among the C24 resistance QTLs, we found dominant, codominant and recessive loci. Interestingly, none of the identified loci significantly contributed to resistance against all three tested Hpa isolates. Our study demonstrates that broad-spectrum resistance of Arabidopsis C24 to Hpa is based on different combinations of multiple isolate-specific loci. The identified quantitative resistance loci are particularly promising as they provide an important basis for the cloning of susceptibility- and immunity-related genes.