The growing demand for sustainable, natural, and health-promoting food ingredients has increased interest in underutilized plant resources, such as Opuntia spp., which are valued for their edible fruits and adaptability to arid environments. Commonly known as prickly pears, these fruits represent a promising raw material for the development of functional foods, being rich in bioactive compounds (e.g., betalains, flavonoids, dietary fibers, essential vitamins, and minerals), which exert antioxidant, anti-inflammatory, and potential antidiabetic effects. Given their capacity to thrive under minimal input conditions in arid and semi-arid climates, Opuntia species align well with sustainability goals in food production. This study evaluates the nutritional potential of eight Opuntia species acclimatized to the temperate continental climate of Cluj-Napoca, Romania, aiming to support functional food diversification and promote the use of climate-resilient crops. Recognizing the central role of soluble carbohydrates in determining fruit quality and nutritional value, this investigation focused on sugar profiling. Ripe fruits were harvested from eight Opuntia spp. accessions maintained in the Succulent Collection of the Agro-Botanical Garden, UASVM Cluj-Napoca. Sugar composition was analyzed using a Shimadzu Prominence isocratic high-performance liquid chromatographic (HPLC) system equipped with differential refractive index detection. Separations were achieved in less than 15 minutes on a Nucleosil amino column, allowing for the simultaneous quantification of glucose, fructose, and sucrose. The results revealed significant interspecific variation in carbohydrate profiles, attributable primarily to genetic differences. Sucrose was predominant in most accessions, with concentrations up to 12 g/100 g and a total carbohydrate content up to 15 g/100 g. By linking biochemical data with phylogenetic relationships, this study provides a basis for selecting Opuntia genotypes suited for food processing. These findings underscore the potential of Opuntia fruits as valuable ingredients in the formulation of valuable functional foods, while also supporting broader objectives related to sustainability, resource efficiency, and climate resilience in food engineering.