Population variation of several microscopic structures used in age-at-death estimation was assessed for three different population samples. The aim of the study was to determine if the need exists for population-specific standards when dealing with individuals of African and European origin. A total sample 223 bone sections from the anterior cortex of the femur (n = 99 black South Africans, n = 94 white South Africans and n = 30 Danish individuals) were analysed using a stereological protocol. Variables assessed included the average number of osteons per grid area (OPD), osteon size and Haversian canal size. ANCOVA was employed for assessment of statistically significant differences. The results indicated that OPD differed significantly between the three groups, but that osteon size was similar for all individuals. Haversian canal size showed unpredictable changes with age and high levels of variation, making it unsuitable to use for age estimation as a single factor. As there are conflicting opinions in the literature on whether to use population-specific equations for the estimation of age-at-death or not, this paper provided additional insight into the use of specific variables and its related variation between groups.