It is predicted that, during the next decades, bioengineering will generate a range of novel tobacco traits intended to add value to the production process or the final product. The first wave of these products is now entering the marketplace, precipitating the immediate need for analytical tools to detect these novel varieties, to differentiate among them, and to distinguish them from conventional varieties. Genetic testing methods, primarily based on the polymerase chain reaction (PCR), have been developed for many agricultural products, such as soy and maize. Application of similar strategies to tobacco has lead to the rapid emergence of effective methods for (1) detecting transgenic tobacco varieties, (2) distinguishing among these varieties, and (3) determining the proportions of different varieties present in a shipment. We will discuss methodological considerations relevant to applying PCR to tobacco analysis, including, (1) sampling, (2) DNA extraction, (3) PCR amplification of target sequences (detection, identification, and quantification), and (4) interpretation of results. We will also discuss issues related to method development, method validation, and quality control. We will conclude with a discussion of future directions in GMO analytical technology.