Root branching order supports a powerful approach to understanding complex root systems; however, how the pattern of root morphological characteristics, tissue carbon (C) and nitrogen (N) concentrations, and root lifespan are related to anatomical features of variable root orders for mature shrubs (∼19 years old) in sandy habitats is still unclear. In this study, these relationships were investigated for three typical shrubs in Horqin Sand Land, Northeast China. Root diameter, individual root length, tissue carbon concentration, C:N ratio, root lifespan, root cross-sectional area (CSA), stele CSA, proportion of stele in root CSA, mean xylem vessel CSA and the number of xylem vessels all increased with root order for the three shrubs, while specific root length and nitrogen concentration decreased with root order. The combined root biomass of the first two orders accounted for more than 63% of the firstfourth order root biomass for all the three shrubs. Proportion of stele to root CSA and number of xylem vessels of third-order root segments were significantly higher than that of the first two orders, and third-order roots showed secondary development with a continuous cork layer. All first-order and most second-order roots exhibited primary development, had an intact cortex, a lower proportion of stele to root CSA, and a smaller number of vessels. Our research suggests that the first two order roots of shrubs in sandy habitats are responsible mainly for absorption, and that they play a major role in root turnover and C and N flux in the soil organic matter pool due to their high proportion of biomass and N concentration, as well as their short lifespan.