Tobacco mosaic virus (TMV) is an extensively studied RNA virus that reduces quality and yield in commercially grown tobacco (Nicotiana tabacum L.). The virus is transmitted mechanically, although infections have been associated with contaminated seeds with the seed coat being the source of virus. Thus, TMV transmission is said to be seedborne (as opposed to true seed transmission where the embryo is infected). The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected K 326 flue-cured cultivar tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was developed to quantify viral titers in each component, while endpoint PCR confirmed RT- qPCR results and established a threshold viral cycle (Ct) value. Endpoint PCR results revealed viral accumulation in all three components of a tobacco seed. The highest concentration of TMV was in the SC, followed by ED and EM. A similar viral concentration gradient was observed in each individual tobacco seed from all three experimental plants. This is the first detection of TMV in tobacco embryos and suggests the virus can be seed transmitted. / Master of Science / Tobacco mosaic virus (TMV) is a widely studied plant virus that affects tobacco, tomato, pepper, and many other crops throughout the world. The virus is easily transmitted through contaminated tools or machinery, workers’ hands or clothing, or when an infected leaf comes into contact with an uninfected leaf. For years, TMV transmission was said to be seedborne, not seed transmitted, meaning that seedling infection comes from the infected external seed coat of the seed. Seed transmission of the virus has yet to be proven because of the difficulty to fully separate tobacco seed tissues. The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample, and synthesized into cDNA for analysis. A quantitative real-time polymerase chain reaction (RT-qPCR) assay was developed to quantify viral concentrations in each component. Endpoint PCR was used to confirm the quantitative results of RT-qPCR. Results revealed TMV accumulation in all three components of a tobacco seed, the highest concentration detected in the SC, followed by ED and EM; this pattern was observed from each plant. This is the first report of TMV being detected in embryos of tobacco seed which suggests that TMV can be seed transmitted.