Tobacco mosaic virus (TMV) causes significant economic losses in production of commercial flue cured tobacco in Zimbabwe. TMV is transmitted mechanically or by any other means that result in the virus coming into contact with injured cells in host plants. Methods such as enzyme linked immunosorbent assay, double stranded RNA analysis, symptomatology and reverse transcriptase PCR have used for detection of TMV. A study to develop a real time quantitative PCR method for the detection and identification of TMV was carried out. Total RNA was extracted and cDNA synthesized using Revert Aid M-MuLV reverse transcriptase enzyme. TMV specific primers and probes were also designed using the Oligo Architect design tool. Using tenfold serial dilutions of standards standard curve qPCR technique was used to estimate viral load in samples. The samples were estimated to contain 1.0002×104 and 1.0003×104 viral copies of TMV/µl. This real time quantitative PCR method provides a quantitative assay which can be used to correlate to disease severity. This assay method could help accurately determine the TMV content of in tobacco.