The Tobacco mosaic virus (TMV) is a positive sense single stranded RNA virus and is found across the world. TMV can impact the overall yield and quality of the crop resulting in an economic loss. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, mottling, necrotic lesions and stunted growth. Historically, TMV has caused controversy on whether this economically significant virus is seedborne or seed transmitted. The objective of this study is to track TMV infection from infected seeds to seedlings to determine the percentage of seed transmission. This experiment used three pods from three different TMV infected cultivar K 326 flue-cured tobacco plants. Seeds from each pod were germinated in a growth chamber for approximately ten days. Samples were separated into seed coat, root and leaves after germination. Total RNA was extracted from each part and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample. Endpoint RT-PCR was used to determine a conservative threshold value from the RT-qPCR results. These results demonstrated that TMV influenced percent germination with a range from 94% to 50%. Seed coats had a significantly higher virus titer concentration (P < 0.05) when compared to the roots and leaves. Statistical analysis revealed highly significant (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Results demonstrate that TMV is seed transmitted in flue-cured tobacco.