Basil downy mildew (BDM) caused by the oomycete pathogen Peronospora belbahrii, threatens sweet basil (Ocimum basilicum) production worldwide. Chemical and cultural control options for BDM are limited, and resistant cultivars have only recently become available for commercial production. To address this challenging agricultural disease, this research investigates BDM epidemiology, occurrences of new pathotypes, and molecular plant-pathogen interactions leading to host resistance or susceptibility. A reproducible low-resource inoculation protocol was developed to harvest P. belbahrii inoculum and propagate BDM for further research. The survival of P. belbahrii sporangia was examined using an in vitro assay to assess germination potential and a plant infection bioassay to determine infection potential following exposure to two temperatures and two humidity levels. Detached P. belbahrii sporangia remained viable up to 12 days when kept isolated at 20ºC in 96.5% relative humidity, significantly longer than had previously been reported. The inoculation procedure was utilized to collect unique P. belbahrii isolates and perform the first controlled-environment differential cultivar trials to identify new pathotypes of BDM. Two new and distinct pathotypes of BDM were identified, one overcoming cultivars with quantitative resistance, and the second overcoming qualitative resistant ‘Prospera’ lines. Interestingly, quantitatively resistant ‘Mrihani’ and ‘Passion’ were more resistant to the second pathotype, indicating that this pair may hold a unique and important source of quantitative resistance. The mechanisms of quantitative disease resistance were explored to identify specific genes as molecular markers for breeding using comparative transcriptomics. Candidate gene MRI-R1 was significantly upregulated in BDM-resistant ‘Mrihani’ following pathogen inoculation and is a member of the canonical resistance gene NB-LRR family. Sequencing of MRI-R1 clones identified four alleles, and two of the identified alleles were isolated from two resistant offspring from the genetic cross using ‘Mrihani’ and BDM-susceptible ‘Newton’ as breeding parents. MRI-R1 likely contributes to quantitative resistance against BDM, and further studies will determine the utility of this gene as a breeding marker. Taken together, these studies have added to the current understanding of BDM epidemiology, the identification of new pathotypes overcoming host resistance, and the identification of candidate genes and mechanisms of quantitative resistance.