The serotonin transporter promoter length polymorphism (5-hydroxytryptamine transporter length polymorphism, 5-HTTLPR) in serotonin transporter gene has been implicated in numerous psychiatric disorders. Having a high affinity for the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT), serotonin transporter controls the duration, availability and signaling capacity of 5-HT in the synapse. Association studies have focused extensively on this polymorphic region as the frequencies of long- and short-alleles of this gene differ greatly amongst populations and association studies have either reported conflicting results or nothing significant at all. In this study, the genotype and allele frequencies of 5-HTTLPR polymorphism were determined in the healthy South African (SA) individuals belonging to diverse ethnic backgrounds. Cheek cell samples were collected from the three major ethnic groups namely: Caucasians, Africans and coloreds/Mixed population. The DNA was extracted and genotyped for the 5-HTTLPR. Genotypes were compared amongst the three major ethnic groups from SA as well as to that of other studies around the world. This is the first study to report significant differences in the 5-HTTLPR genotype and allelic frequencies among various ethnic groups in SA. Future studies will target larger population groups and the estimation of frequency of these alleles in individuals with autism.