Black shank is typically the most important disease affecting tobacco in the United States and is caused by the soil-borne oomycete Phytophthora nicotianae. Genetic resistance is an essential component of overall strategies to reduce economic loss from disease. Polygenic resistance derived from cigar cultivar ‘Florida 301’ provides high levels of partial resistance to all races of P. nicotianae. Most U.S. tobacco cultivars possess varying levels of resistance derived from this source material. Identification of DNA markers associated with genomic regions controlling resistance in Florida 301 could facilitate development of resistant varieties in the future. A recombinant inbred line (RIL) population consisting of 122 F_5:6 lines was produced from a cross between ‘Florida 301’ and the black shank-susceptible cultivar ‘Hicks’ and evaluated for black shank resistance in three field environments and a single greenhouse experiment. The population was also genotyped at a total 339 polymorphic microsatellite marker loci. QTL analysis resulted in the identification of 11 QTL affecting area under disease progress curve (AUDPC) in the field evaluation, and 11 QTL affecting resistance in the greenhouse experiment. The most significant QTLs were important in both the field and greenhouse experiments. The QTL on PMI linkage group 7 was found to have the largest effect in the Florida 301 × Hicks RIL population. This QTL was also found to be the most important in a previously published experiment using a Beinhart 1000 × Hicks doubled haploid population. The large QTL on PMI LG 15 found to be important in the latter population was not found to be significant in the current population, thus suggesting the uniqueness of this genetic variability to U.S. flue-cured and Burley tobacco. Consistent with previous publications, the inheritance of the ‘Florida 301’ type of black shank resistance is likely complex.