Phytophthora infestans, the causal agent of late blight, remains the main threat to potato production worldwide. Screening of 19 accessions of Solanum dulcamara with P. infestans isolate Ipo82001 in detached leaf assays revealed strong resistance in an individual belonging to accession A54750069-1. This plant was crossed with a susceptible genotype, and an F(1) population consisting of 63 individuals was obtained. This population segregated for resistance in 1:1 ratio, both in detached leaf assays and in an open-field experiment. Presence of the formerly mapped Rpi-dlc1 gene as the cause of the observed segregating resistance could be excluded. Subsequently, AFLP analyses using 128 primer combinations enabled identification of five markers linked to a novel resistance gene named Rpi-dlc2. AFLP markers did not show sequence similarity to the tomato and potato genomes, hampering comparative genetic positioning of the gene. For this reason we used next-generation mapping (NGM), an approach that exploits direct sequencing of DNA (in our case: cDNA) pools from bulked segregants to calculate the genetic distance between SNPs and the locus of interest. Plotting of these genetic distances on the tomato and potato genetic map and subsequent PCR-based marker analysis positioned the gene on chromosome 10, in a region overlapping with the Rpi-ber/ber1 and -ber2 loci from S. berthaultii. Pyramiding of Rpi-dlc2 and Rpi-dlc1 significantly increased resistance to P. infestans, compared with individuals containing only one of the genes, showing the usefulness of this strategy to enhance resistance against Phytophthora.