Abstract: Burley tobacco variety B21 and Maryland tobacco variety Md609 were used as experimental plants, the contents of important precursors in the biosynthesis of chlorophyll and the activities of key enzymes in leaves of the two varieties were compared to reveal the physiological and biochemical mechanisms of low chlorophyll content in burley tobacco leaves. The results showed that the contents of precursors of chlorophyll biosynthesis, δ-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrinogen Ⅲ (Urogen Ⅲ), coproporphyrinogen Ⅲ (Coprogen Ⅲ), protoporphyrin Ⅸ (Proto Ⅸ), Mg-protoporphyrin Ⅸ (Mg-Proto Ⅸ) and protochlorophyllide (Pchlide), in the leaves of B21 were lower. The activities of 5-aminolevulinate dehydratase and porphobilinogen deaminase, which catalyzed the synthesis of chlorophyll, in the leaves of B21 were 0.43% and 0.75% of those of Md609, respectively; while the activity of chlorophyllase, which catalyzed the degradation of chlorophyll, in leaves of B21 was 2.04 times the activity of that in Md609. It indicated that the low chlorophyll content in burley tobacco might be the results of the inhibited synthesis and accelerated degradation of chlorophyll in leaves.