A novel analytical method for the determination of chloranisoles (2,4-dichloroanisole, 2,6-dichloroanisole, 2,4,6-trichloroanisole and 2,3,4,6-tetrachloroanisole) in tobacco was developed by using gas chromatography-tandem mass spectrometry (GC-MS/MS). In this method, Concurrent Solvent Recondensation Large Sample Volume Injection (CSR-LVI) was applied using a temperature-programmed inlet, and by applying a large injection volume of 25 µL, which greatly enhanced the analytical sensitivity of ultra-trace chloranisoles. Florisil SPE cleaning of tobacco sample extracts was performed for the sample pre-treatment, followed by a back-flush technique to expel the high boiling point components, reducing the contamination of the chromatographic system. This method exhibited ultra-high sensitivity, excellent selectivity, recovery and repeatability, and was suitable for routine analysis of multitudinous tobacco samples. The method was applied to investigate changes in the contents of chloranisoles and in the musty odour of flue-cured tobacco during the mouldy tobacco cultivation process. The result showed that the contents of chloranisoles, especially of 2,4-dichloroanisole and 2,4,6-trichloroanisol, was correlated with the degree of musty odour, and a quantitative relationship between two chloranisoles and musty odour in tobacco could be established.