In Nicotiana tabacum L., four major alkaloids, including nicotine, are synthesized. It has been known for some time that two independent loci, Nic1 and Nic2, are key biosynthetic regulators and that the effect of the Nic1 locus is about two-fold greater than the effect of the Nic2 locus on nicotine levels. Although, seven ERF genes within the Nic2 locus were previously characterized, little is known about the Nic1 locus. In this study, we focused on identifying the Nic1 locus in Burley tobacco. The TN90 genome was sequenced. To identify the Nic1 locus, the near isogenic lines (NILs), Burley 21 (BU21, Nic1Nic2), High Intermediate (HI, Nic1nic2), Low Intermediate (LI, nic1Nic2) and Low Alkaloid BU21 (LA, nic1nic2), were also sequenced. By comparing sequencing data of the 4 NILs to TN90, we identified a putative Nic1 region. To improve elucidation, sequencing was also performed on bacterial artificial chromosomes created from tobacco genomic DNA. We have now identified a large chromosomal region that spans both borders of the Nic1 locus. Primers for genes within Nic1 locus were tested and validated along with known Nic2 ERF189 primers on two F2 populations (~510 individuals each) segregating for nic1 and nic2 loci. Genotypic segregation ratios of 9:3:3:1 as well as alkaloid levels consistent with identified genotypes were observed. Identification of the Nic1 genomic location facilitates not only characterization of its functional genes but also development of SNP markers to enable high throughput screening and identification of low alkaloid traits in breeding populations.