Opuntia ficus-indica (L.) Mill. is one of the most recognisable agricultural crops that can withstand harsh environmental conditions. South Africa is one of the few countries that hosts a large germplasm of Opuntia cultivars, which represent an ex-situ conservation population. However, little is known about the genetic diversity in this population. Additionally, some genotypes are morphologically indistinct, and therefore, making it a challenge for novice farmers and researchers to recognise specimens in the germplasm. The current study aimed to differentiate and measure the genetic diversity in 44 cultivars that represent the South African Opuntia germplasm using eight simple sequence repeat (SSR) markers. Evidently, the cultivars comprised moderate levels of diversity (average polymorphic information content, PIC = 0.37, Nei’s unbiased gene diversity = 0.42) that discriminated 90% of the cultivars. Analysis of the cultivars with the unweighted pair-group method using arithmetic averages (UPGMA) method revealed three main clusters, whereas principal coordinate analysis (PCoA) showed indistinct clustering of cultivars based on their usage in the agricultural market. Overall, the used SSR markers were able to distinguish most cultivars in the South African Opuntia germplasm—a genetic resource that appears to hold sufficient genetic diversity to aid the conservation and breeding of novel cultivars for the agriculture market.