To determine the meaning of graylevels in backscattered electron (BSE) images of actual bone tissues, the influence of mineral content and mineral composition on BSE image graylevels was studied using chick bone tissue representing a broad age range. These tissues were analyzed for BSE image graylevels, Ca/P molar ratios, mineral composition, mineral content (v/v), ash fraction (w/w), and density (g/cm³). Linear regression analyses showed that the weighted mean graylevels (WMGLs) in BSE images were positively correlated to ash fraction (r² = 0.711), mineral content (r² = 0.720), and density (r² = 0.843). Although the Ca/P ratio increased from 1.65 in embryos to 1.80 in 2‐year olds, the compositional changes corresponding to this Ca/P molar ratio were estimated to produce a relatively minor (< 4.0%) change in BSE image graylevel. These results demonstrate that graylevels in BSE images of actual bone tissue can be attributed to mineral content and density, but only as a coincidence of their association with atomic number.