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.