The oomycete Phytophthora nicotianae is one of the most economically important pathogens affecting tobacco (Nicotiana tabacum L.). Genetic resistance is a preferred means of managing damage caused by Phytophthora, and genes conferring partial resistance are likely to be more durable over time than those controlling immunity. Characterization of specific genetic variability controlling partial disease resistance may aid the development of long-term strategies for minimizing crop plant disease loss. Previous mapping studies identified a quantitative trait locus (QTL) designated as Phn7.1 controlling partial P. nicotianae resistance. Favorable alleles at this QTL have been identified in cigar tobacco cultivars ‘Beinhart 1000’ and ‘Florida 301,’ and were thought to also be present in most modern elite US flue-cured tobacco germplasm. To gain increased insight of the location and effect of Phn7.1, this QTL was mapped using an increased number of molecular markers (SNPs) in the genomic region of interest. A series of near isogenic lines (NILs) and sub-NILs carrying the Phn7.1-associated genomic region introgressed from Beinhart 1000 in the genetic background of susceptible variety Hicks were developed and evaluated. The region was found to have an additive effect on resistance and the corresponding QTL was localized to within a genetic interval of approximately 3 cM. Genotyping of historical materials with Phn7.1-associated SNP markers strongly suggests that the favorable Phn7.1 allele(s) is present in most modern US flue-cured cultivars and absent in early predecessor germplasm. Information from this study may be useful in marker-assisted selection and for identification of Phn7.1 candidate genes in future investigations.