Sclerotinia sclerotiorum (Lib.) de Barry, commonly known as Sclerotinia stem rot is one of the pathogens that have a potentially devastating impact on the growth of soybean industry in South Africa. Several quantitative trait loci (QTL) that play a role in soybean resistance to Sclerotinia stem rot have been identified and mapped on the soybean’s integrated genetic linkage map. However, none of these QTL and their underlying markers have been evaluated in any of the commercial soybean cultivars grown in South Africa. The aim of this study was to screen and characterise SSR markers linked to QTL that are reported to have an association with stem rot resistance on South African commercial soybean [Glycine max (L.) Merr] cultivars. Twenty SSR markers that have been reported to have an association with stem rot resistance on soybean were evaluated in 29 commercial soybean cultivars that are currently planted in South Africa. The genetic diversity of markers and the genetic relationships across the cultivars were evaluated using the GenAlEX software package. Five markers were not polymorphic, while twelve markers were polymorphic across all cultivars. Evaluation of genetic relationships across cultivars showed that 10 South African cultivars share alleles with a cultivar with known resistance (M.A) to Sclerotinia stem rot. We propose that the shared alleles between 10 South African cultivars and M.A could indicate shared resistance to Sclerotinia stem rot and indicates the presence of a genetic rather than environmental mechanism of resistance in South African soybean cultivars. The results from this study will aid South African soybean breeders in knowing which markers they can use to screen for Sclerotinia stem rot resistance within the context of South African soybean cultivars.