Rdrl is the first monogenic dominant resistance gene described in the genus Rosa that is active against black spot of roses caused by Diplocarpon rosae, a facultative biotrophic parasitic ascomycete. It was introgressed from the wild rose species Rosa multiflora. According to the previous studies, the Rdr1 resistance locus carries nine copies of resistance gene analouges (RGA) of the Toll-interleukin 1 receptor (TIR), nucleotide binding site (NBS), leucine rich repeat (LRR) type within an interval of 220Kb of DNA. In the present study, the functional characterization of Rdr1 candidates led to the identification of a single major resistance gene active against black spot of roses. Initial sequence analysis, generation of single RGAs and homologous and heterologous expression studies of Rdr1 candidates reduced the number of candidate genes to five. One of the remaining five candidates, RGA8 could restrict the growth of Dort E4 significantly when used to complement the susceptible rose variety transiently. Transient homologous disease assay proved RGA8 as the single major resistance gene that confers resistance to black spot in roses.