Abstract Muscadines face limitations to fresh market production due to high manual labor costs. Mechanical harvesting holds promise for reducing the costs associated with muscadine production but requires cultivars with easily detached fruit at maturity. This study aimed to determine muscadine fruit and pedicel characteristics influencing fruit detachment force (FDF) and to unravel the genes, hormones, and regulatory networks governing muscadine abscission. We characterized the FDF of muscadine fruit across 18 genotypes and at four developmental stages. Following this, we performed a transcriptome analysis using the mature pedicel tissue of two genotypes, a genotype with high FDF at maturity and a genotype with low FDF at maturity, to identify differentially expressed and uniquely expressed genes contributing to fruit detachment. We found that pedicel length, pedicel–fruit junction area, and fruit diameter positively correlated with FDF. This study also identified novel candidate genes, transcription factor families, and pathways associated with muscadine fruit abscission. These findings provide valuable knowledge on the progression of fruit abscission and insights for reducing FDF, particularly in developing machine-harvestable muscadine cultivars and fostering sustainability and efficiency in muscadine production.