Nicotine is the most abundant alkaloid in cultivated tobacco (Nicotiana tabacum), typically constituting more than 90% of total alkaloids. Recently, the U.S. Food and Drug Administration (FDA) issued an Advance Notice of Proposed Rulemaking (ANPRM) to obtain information for consideration in developing a tobacco product standard to set the maximum nicotine level in cigarette filler to “minimally addictive or non-addictive” levels. Nicotine levels are highly variable across different years and locations as data collected over decades in the Minimum Standards Programs show. Analysis of the Burley check varieties in the Minimum Standards Program showed relative standard deviations ranging from 20% to 28% for individual varieties across locations from 2012 to 2018. Though there are decades of nicotine analysis from the Minimum Standards Program, there is not much data collected and publicly available on nicotine variability in low nicotine cultivars. The purpose of this study is to determine variability in nicotine content across multiple years and locations from low nicotine cultivars and compare it to normal nicotine cultivars. To initiate this long term study we analyzed low nicotine cultivars alongside normal nicotine cultivars at the same locations from 2012 to 2018. Assuming the variability in nicotine content for low alkaloid cultivars is similar to or greater than the variability in normal nicotine cultivars, it will have major impacts on meeting any regulatory standard. This could suggest that to meet a standard of 0.3-0.5 mg/g nicotine content the cultivars themselves may need to have much lower nicotine levels to consistently meet the proposed standard year after year. This will have major implications on the technical achievability of such a standard.