Background Trichomonas vaginalis is the causative agent of trichomoniasis. The genetic characterisation of T. vaginalis isolates reveals significant genetic diversity in this organism. Data on the prevalence of different genotypes of T. vaginalis in South African populations is lacking. This study investigated the diversity of T. vaginalis in a pregnant population in South Africa. Methods In this study, 362 pregnant women from the King Edward VIII Hospital in Durban, South Africa, provided vaginal swabs to be tested for the presence of T. vaginalis. T. vaginalis was detected using the TaqMan assay using commercially available primers and probes specific for this protozoan (Pr04646256_s1). The actin gene from T. vaginalis was amplified with gene-specific primers. The actin amplicons were digested with HindII, MseI, and RsaI, and the banding patterns were compared across the three digests for assignment of genotypes. Phylogenetic analysis was conducted using MEGA. Results The prevalence of T. vaginalis in the study population was 12.9% (47/362). Genotype G was the most frequent genotype in our study population. Genotypes H and I were detected in one sample each. According to the multiple sequence alignments and phylogenetic analysis, a level of diversity was observed across and within genotypes. Four different single-nucleotide changes in the actin gene were detected. Sample TV358 (H genotype) contained a single amino acid substitution from glutamine to lysine. Sample TV184 (G genotype) contained a single amino acid substitution from glutamic acid to arginine. Sample TV357 (G genotype) contained two amino acid substitutions, arginine to leucine and glycine to aspartic acid. Conclusion Three different genotypes were observed in the pregnant population. Diversity was observed across and within genotypes. The observed diversity can be challenging for future vaccine design and development of antigen-based rapid diagnostic tests for trichomoniasis.