Oral mucosal lesions (OML) can decrease oral health-related quality of life and some have the potential to become malignant. The aim of the present study was to report the prevalence of OML in relation to age, sex, and serum cotinine levels in a population with mixed ancestry from South Africa. This study is part of the Cape Town Vascular and Metabolic Health (VHM) study, conducted between 2014–2016. Trained dental examiners assessed the oral mucosa for the presence of OML according to WHO criteria. In total, 1976 individuals were included in the study, being 1496 females (75.7%) and 480 males (24.3%) with average age of 49.5 years (SD = 15.3). In total, 262 lesions were detected in 252 participants (overall prevalence of 13%). Males had higher prevalence than females (14% vs. 9%, p = 0.008). Participants aged 25–34 had the highest prevalence rates (21%). Participants who had cotinine ≥15 ng/mL had higher prevalence of OML as compared to those with <15 ng/mL (15% vs. 5%, p < 0.001). Most common lesions were nicotine stomatitis (33%) and leukoplakia (19%). Age, male sex, and higher cotinine levels were associated with increased prevalence of OML.