The Tobacco mosaic virus (TMV) has infected tobacco plants since the late 1800’s, causing detrimental yield and economic losses in tobacco. TMV is classified as a seed borne virus because the virus infects tobacco seed as a contaminant on the seed coat surface. The purpose of this study is to investigate seed-borne transmission of Tobacco mosaic virus (TMV) by examining the infestation route of tobacco seeds. Four different crosses were performed using K326 flue-cured variety: 1) self-fertilized, TMV infected, 2) self-fertilized, non-TMV infected, 3) TMV maternal infected, and 4) TMV paternal infected. Tobacco seeds were collected from three individual pods for each cross. Total RNA was extracted from 100 individual seeds per pod, and synthesized into cDNA for analysis. A quantitative real-time PCR (qRT-PCR) assay was developed to analyze TMV concentrations within individual tobacco seeds. qRT-PCR was adopted over other traditional viral detection methods for its capability of generating fast quantitative results in real time. Results revealed distinct TMV concentration patterns and data suggests uneven distribution of TMV within individual seed pods. These results show evidence of maternal but not paternal seed-borne transmission of TMV. Furthermore, dissection of individual seed reveals that the majority of TMV found in the seed is located within the seed coat and not the embryo. These findings may be useful in identifying the TMV infection route of entry in emerging tobacco seedlings.