Fragmented human remains present a challenge for forensic experts as they attempt to identify individuals using standard forensic methods. Several histological age estimation techniques have been developed during the last fifty years to aid in this process. However, very few validation studies have been conducted in order to test their accuracy and bias, and thus, validation assessment is required as we employ them while testifying in court. Histological variables are assessed from rib thin sections from two Mediterranean samples; Cretans (N = 41) and Greek-Cypriots (N = 47). Intra and inter-observer errors are assessed through TEM analysis and Intra-class Correlation Coefficient by testing observers with different levels of experience as they collected data on osteon counts and area measurements. The relation between the variables and age is determined using correlation coefficients. Histomorphometric data are applied to four widely used age estimation formulas assessing the performance of the methods for the entire sample. Inaccuracy and bias are calculated with age estimations and known age tested for significance and proportional bias assessed. Overall, histological parameters presented acceptable intra- and inter-observer errors. All variables exhibited statistically significant correlation with age (P < 0.01). For three of the techniques, data showed a systematic underestimation of age with an increase in inaccuracy in older individuals. One of the age estimation formulas produced overestimation of young individuals yet, it more accurately estimated the age of older individuals. This validation study explores inter-population variation in bone remodeling dynamics and presents a critical evaluation on methodological issues that can affect the performance of existing histological techniques.