Non-structural carbohydrates (NSC) are important carbon reserves in trees, which vary seasonally due to changes in the strength of carbon sink or source. Seasonal fluctuations of NSC in branches are influenced by multiple factors such as leaf phenology, shoot extension, and radial growth, but it is not clear which factor plays the key role. Here we measured the NSC concentrations in the new twigs (current year) and old branches (≥ 1 years old, diameter < 3 cm) at seven phenological stages for 12 temperate tree species in northeastern China. The objectives were to (1) compare seasonalities of NSC concentrations in the new twigs and old branches, (2) explore relationships between concentrations of soluble sugars and starch in these tissues, and (3) explore factors controlling the inter-specific variations in NSC concentrations in branches. The species included three evergreen conifers[Korean pine (Pinus koraiensis), Mongolian pine (Pinus sylvestris var. mongolica) and Korean spruce (Picea koraiensis)], one deciduous conifer[Dahurian larch (Larix gmelinii)], and eight deciduous broadleaved species[Mongolian oak (Quercus mongolica), Japanese elm (Ulmus japonica), Manchurian ash (Fraxinus mandshurica), Manchurian walnut (Juglans mandshurica), Korean aspen (Populus davidiana), Ussuri poplar (Populus ussuriensis), white birch (Betula platyphylla), and Amur linden (Tilia amurensis)]. For each species, one branch at the mid-crown was sampled and separated into new twigs and old branches. Concentrations of soluble sugars and starch were determined with a modified phenol-sulphuric method, and expressed as a percentage of dry matter (% DM). The concentrations of NSC in new twigs and old branches showed similar seasonal patterns of NSC concentration for all the tree species except for Korean aspen and Ussuri poplar. The concentrations of NSC in the new twigs were usually higher than those in the old branches. The concentrations of TNC (total non-structural carbohydrates, sum of soluble sugars and starch) in the old branches of the evergreen species increased significantly before bud-break, declined slightly during bud-break, and kept relatively stable after leaf flushing until autumn when the concentration of NSC rebounced slightly. The concentrations of TNC in the old branches of the deciduous species decreased significantly before or during bud-break, and then progressively increased in both new twigs and old branches along the season. Meanwhile, a conversion of starch to soluble sugars was observed in October when the dormant season started. There was a significant and positive linear relationship between the concentrations of soluble sugars and starch for both new twigs and old branches when the data were pooled across all the species. The seasonal mean concentration, maximum concentration, and storage capacity of TNC in the old branches decreased significantly with the duration of shoot extension increasing for all the species except for Mongolian oak (the species with an intra-annual regrowth of branch), Korean spruce and Japanese elm (no data available). These findings suggested that the shoot growth of these temperate trees relies strongly on NSC reserves, and the duration of shoot length growth is inter-connected with TNC storage in branches. Future researches on forest carbon cycle modeling and global climate change should consider this functional inter-connection.