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In higher plants, vacuolar invertases play essential roles in sugar metabolism, organ development, and sink strength. In sorghum (Sorghum bicolor), two vacuolar invertase genes, SbVIN1 (Sobic. 004G004800) and SbVIN2 (Sobic. 006G160700) have been reported, but their enzymatic properties and functional differences are largely unknown. We combined molecular, biochemical and genomic approaches to investigate their roles in sorghum stem and grain traits. SbVIN1 and SbVIN2 showed different expression levels in internodes, leaves, and panicles, indicating that their importance in each organ was different. In an in vitro sucrose hydrolysis assay, proteins of both genes heterologously expressed in Pichia pastoris displayed similar enzyme properties including the same optimum reaction pH (5) and similar Km for sucroe (49 mmol L−1 and 45 mmol L−1 for SbVIN1 and SbVIN2, respectively). The optimum reaction temperatures of SbVIN1 and SbVIN2 were 45 °C and 65 °C, respectively. SbVIN2 showed higher tolerance to high temperature than SbVIN1. We characterized the sequence variation of these two vacuolar invertase genes in a panel of 216 diverse inbred lines of sweet and grain sorghum and performed gene-based association analysis. SbVIN1 showed significant associations with stem traits including stem length, stem diameter, internode number, stem fresh weight, and Brix, as well as grain traits including hundred-grain weight and grain width. Significantly associated variation sites were mainly in 5′ upstream and intron regions. SbVIN2 only associated with grain width and stem water-soluble carbohydrates (WSCs) content. We conclude that the vacuolar invertase genes SbVIN1 and SbVIN2 are differently associated with stem and grain traits in sorghum. Keywords: Sorghum (Sorghum bicolor), Vacuolar invertase, SbVIN1, SbVIN2, Enzymatic properties, Association analysis