This is the first extensive study of a large sample of ancient Greek dentition (351 individuals with over 4500 teeth and over 5500 alveoli) from one location, the Greek colony of Metapontion (modern Metaponto) in Southern Italy, and the only dental study exploring rural and urban biological differences within one Greek colony. The impact of rural and urban lifestyles on dental health is described with the frequency of caries, hypoplasia, abscesses, periodontal disease, and dental wear. Biological distances between urban and rural Metapontines and other world populations are studied using metric (tooth diameters) and 44 non-metric dental traits such as Carabelli’s cusps, peg shaped incisors, cusp number and occlusal surface patterns on molars, etc. Biological influence of Greek colonists on the indigenous Italic population is discussed. Dental traits are also used to confirm the existence of family plots within burial grounds.