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Abstract Biomass microbiota and chemical constituent are closely associated with final anaerobic fermentation performance. But the limiting factors affecting anaerobic fermentation quality and bacterial community have been rarely explored. This study aimed to elucidate the relative contribution of initial microbiota and chemical constituent of sweet sorghum on its final anaerobic fermentation quality. Sweet sorghum at two developmental stages (heading-stage, G1; hard-dough-stage, G2) was treated as follows: G1 microbiota + sterilized G1 (M1C1), G2 microbiota + sterilized G1 (M2C1), G1 microbiota + sterilized G2 (M1C2), and G2 microbiota + sterilized G2 (M2C2). The results showed that chemical constituent rather than microbiota changes remarkably influenced the production of lactic acid, propionic acid and ammonia-N, the relative abundance of Lactobacillus, Weissella, Lactococcus, Pediococcus, and Pantoea of sweet sorghum after anaerobic fermentation. The chemical constituent was the key limiting factor affecting the anaerobic fermentation quality of sweet sorghum. This study could provide a reference for clarifying the key limiting factors affecting anaerobic fermentation and making recommendations for production. Graphical Abstract