Muscadines (Muscadinia rotundifolia Michx) are native to the southeastern United States and have been cultivated for fresh consumption, juice, wine, and other processed products for over 100 years. Manual harvesting is one of the main challenges for fresh market production due to high labor costs. This study aimed to determine muscadine fruit and pedicel characteristics influencing fruit detachment force and to unravel the genes, hormones, and regulatory networks governing muscadine abscission. We characterized the fruit detachment force of muscadine berries across eighteen 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 fruit detachment force at maturity and a genotype with low fruit detachment force at maturity, to identify differentially expressed and uniquely expressed genes contributing to fruit detachment. Our results suggest that the abscission process and abscission zone development likely occur at different rates in a genotype and developmental stage dependent manner. Our findings provide valuable knowledge on the progression of fruit abscission and insights for controlling fruit detachment force. This will aid in the development of machine-harvestable cultivars which will increase the sustainability and efficiency of muscadine production.