<p>Sugar metabolism is a central determinant of grape berry quality, influencing sweetness, texture, and consumer preference. Comparative insights into enzymatic and transcriptional regulation of sugar accumulation among muscadine (<i>Vitis rotundifolia</i> Michx), hybrid bunch (<i>Vitis</i> spp.), and bunch (<i>Vitis vinifera</i> L) grapes remain limited. We examined: four representative genotypes two muscadines (Black Beauty, Regale), one hybrid bunch (Blanc du Bois), and one bunch grape (Barbera) across four developmental stages: pea, green, veraison, and ripe. Berry weight and diameter increased progressively, with muscadines producing the largest berries, particularly Black Beauty (~ 13.1&#xa0;g; 29.6&#xa0;mm). Total soluble solids rose from 4.0 to 15.8 °Brix, whereas titratable acidity declined during ripening. Sucrose accumulated exclusively in muscadines at veraison and ripening, while glucose and fructose dominated all genotypes and rose sharply at ripening, with total sugar concentration ranking Barbera &gt; Blanc du Bois &gt; Black Beauty &gt; Regale. Muscadines exhibited enhanced sucrose synthesis and retention, reflected by higher sucrose-phosphate synthase and sucrose synthase activities and concomitant invertase inhibition. In contrast, hybrid and bunch grapes favored invertase-driven sucrose cleavage and hexose accumulation. Elevated expression of invertase inhibitor genes (<i>VvInvI15</i>,<i> VvInvI2(1)</i>, and <i>VvInvI18(1)</i>) in muscadines supports post-translational suppression of invertase activity, while reduced <i>VvSWEET15</i> expression suggests lower sugar import capacity during late development. Multivariate analyses, including principal component and heatmap clustering, revealed distinct metabolic and transcriptional signatures separating muscadine from bunch grapes. These findings provide a mechanistic link between domestication and sugar metabolism and highlight key enzymatic and regulatory nodes for improving berry sweetness in grape breeding programs.</p>