High rates of nitrogen fertilization are required currently for cultivation of Burley tobacco to achieve yields and qualities that are desired. However, Maryland type tobaccos are grown using a nitrogen fertilization rate that is ~60-70% lower than that used typically for Burley tobacco. The objective of this study was to identify possible molecular markers that differentiate nitrogen utilization of Maryland tobacco compared to Burley tobacco. Maryland and Burley tobacco plants were grown in the greenhouse using either 100 ppm nitrogen or 25 ppm nitrogen and were fed continuously in an ebb and flow system. Gene expression was analyzed at a whole genome level by RNA-seq. Metabolite profiling was done using multiple approaches. Profiling of these two tobacco types revealed a difference in the carbon-nitrogen balance and an increase in pools of non-essential nitrogen containing compounds in Burley tobacco at low fertilization rates as compared to Maryland tobacco. Genes that may be responsible for these observations were also differentially regulated when compared. Possible molecular markers were measured in hybrid populations resulting from crosses between Maryland and Burley tobacco to validate their use as markers for nitrogen use efficiency. This study not only revealed possible gene targets that could be modified to improve nitrogen utilization in Burley tobacco, but also identified molecular markers that could be used for selecting nitrogen use efficient lines in breeding programs.