South Africa hosts the largest cactus pear germplasm bank in Africa. However, since cactus pear undergoes significant genotype x environment (G x E) interaction, characterisation based on agronomic and morphological traits in a country such as South Africa is complicated by its wide agro-ecological regions. However, the effects of G x E interaction on characterisation can be circumvented by the use of molecular markers. Therefore, AFLP markers were used to estimate the genetic diversity among 38 cactus pear cultivars from the South African genebank. The number of polymorphic fragments between different accessions varied with the use of different AFLP-primer combinations, suggesting that sufficient detectable genetic differences exist within the germplasm for the use of AFLP markers for genetic diversity assessment, cultivar identification and parental selection. A large number of markers had polymorphic information content (PIC) values between 0.3-0.5, indicative of good discriminatory ability. The majority of the cultivars grouped into two main clusters. The commonly grown cultivars were dispersed amongst the different clusters, with the greatest percentage falling in clusters i and sub-cluster iib. Culitvars that originated from Botswana (R1251, R1259, and R1260) clustered together, whilst those from Israel (Sharsheret', 'Ofer', and 'Messina') were dispersed within cluster ii. Ten genotype specific fragments (GSF) were generated from six primer combinations (E-AGG + M-CAT, E-ACT + M-CAG, E-ACT + M-CAT, E-ACA + M-CAT, E-ACA + M-CTT, and E-ACA + M-CAG). These GSF should be tested further, since they will allow varietal identification if proven to be repeatable. The genetic similarity values indicated in this study will provide breeders with information useful for selecting diverse parents with desired traits for their crosses.