Sweet basil (Ocimum basilicum) is susceptible to downy mildew caused by the oomycete foliar pathogen Peronospora belbahrii. No resistant varieties of sweet basil are commercially available. Here, we report on the transfer of resistance gene Pb1 from the highly resistant tetraploid wild basil O. americanum var. americanum (PI 500945, 2n = 4x = 48) to the tetraploid susceptible O. basilicum 'Sweet basil' (2n = 4x = 48). F1 progeny plants derived from the interspecific hybridization PI 500945 × Sweet basil were resistant, indicating that the gene controlling resistance (Pb1) is dominant, but sterile due to the genetic distance between the parents. Despite their sterility, F1 plants were pollinated with the susceptible parent and 115 first backcross generation to the susceptible parent (BCs1) embryos were rescued in vitro. The emerging BCs1 plants segregated, upon inoculation, 5:1 resistant/susceptible, suggesting that resistance in F1 was controlled by a pair of dominant genes (Pb1A and Pb1A'). Thirty-one partially fertile BCs1 plants were self-pollinated to obtain BCs1-F2 or were backcrossed to Sweet basil to obtain the second backcross generation to the susceptible parent (BCs2). In total, 1 BCs1-F2 and 22 BCs2 progenies were obtained. The BCs1-F2 progeny segregated 35:1 resistant/susceptible, as expected from a tetraploid parent with two dominant resistant genes. The 22 BCs2 progenies segregated 1:1 resistant/susceptible (for a BCs1 parent that carried one dominant gene for resistance) or 5:1 (for a BCs1 parent that carried two dominant genes for resistance) at a ratio of 4:1. The data suggest that a pair of dominant genes (Pb1A and Pb1A') residing on a two homeologous chromosomes is responsible for resistance of PI 500945 against P. belbahrii.