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Abstract Understanding the age-structure of mammal populations is important for understanding population dynamics and informing wildlife conservation and management. For decades, cementum annuli analysis (CAA), the process of estimating animal age by counting cementum annuli in teeth, has been a standard method used by wildlife biologists from around the globe. The process of histological preparation for CAA of demineralized teeth often involves multiple steps that can be achieved using a variety of techniques. One technical issue that has been largely unexplored is understanding the effect of tooth section thickness on the process of accurately aging mammals by CAA. In this study, we investigated the relationship between validation of 2 experts aging mammals identically by CAA and tooth section thickness, species, and animal age. Our purpose was to determine if cutting teeth thicker than our standard of 14 µm compromises the ability of experts to age teeth identically. To carry out our study, 2 experts used CAA to determine the age of 30 American Black Bear (Ursus americanus) from Alaska, 30 Elk (Cervus canadensis) from Colorado, and 30 White-tailed Deer (Odocoileus virginianus) from Maine using teeth cut to different section thicknesses: 14 µm (our standard), 25 µm, and 35 µm. Ages ranged from 1 to 8 yr for American Black Bear, 2 to 7 yr for Elk, and 1 to 17 yr for White-tailed Deer. We found that more teeth were aged identically by experts at 14 µm thickness (96%) compared to 25 µm (79%) and 35 µm thickness (82%). Age assignments from the experts agreed most frequently for Elk (93%), followed by American Black Bear (89%) and White-tailed Deer (74%). Animal age had no effect on experts aging teeth identically. Future studies interested in exploring an optimal tooth section thickness for CAA should strive to include individuals spanning the latitudinal range of the species and to use known-age animals whenever possible.