Genetic variants in multiple cellular pathways have been associated with an altered risk of oesophageal cancer. In this study, eight genes previously associated with an altered risk of oesophageal squamous cell carcinoma (OSCC) in European or Asian populations were investigated in two South African populations. We genotyped 12 single-nucleotide polymorphisms and one insertion/deletion variant in 1463 individuals from the Black and Mixed Ancestry populations. No polymorphisms were associated with OSCC in the Black population. In the Mixed Ancestry population, ALDH2 +82 G > A (rs886205) was significantly associated with a reduced risk of OSCC (odds ratio = 0.70, 95% confidence interval = 0.55–0.89; P = 0.0038). Several other polymorphisms showed a suggestive association (P A (rs689466), CASP8 Asp302His (rs1045485) and MGMT Leu84Phe (rs12917). Haplotype analysis indicated that the FAS polymorphisms −670 A > G (rs1800682) and −1377 G > A (rs2234767) were both associated with OSCC in the Mixed Ancestry population (P = 0.006 and P = 0.004, respectively), as well as the CASP8 (−652 6Ndel:302His) haplotype (P = 0.0013). This study indicates several instances of population-specific differences in the genetic etiology of OSCC between these two South African populations and between them and other high-risk populations, which may reflect differences in their ancestry and environmental exposures.