Natural organic matter was collected from the upper Yukon River and size fractionated into the <1 kDa low‐molecular‐weight dissolved (LMW‐DOC), colloidal (COC, 1 kDa to 0.45 μm) and particulate organic carbon (POC, >0.45 μm) phases for characterization of elemental (C and N) and isotopic (13C, 14C and 15N) composition to examine their sources and transport. Concentrations of total organic carbon (TOC) decreased from 3010 μM in mid‐May to 608 μM in September, accompanying an increase in river water δ18O from the snowmelt to summer and early fall. COC was the predominant OC species, comprising, on average, 63 ± 8% of the TOC, with 23 ± 5% partitioned in the LMW‐DOC and 14 ± 5% in the POC fraction. Annual riverine export flux to the ocean was 2.02 × 1012 g‐C for TOC, 7.66 × 1010 g‐N for total organic nitrogen (TON), and 3.53 × 1012 g‐C for dissolved inorganic carbon (DIC), respectively. The C/N molar ratios were distinctly different between colloidal organic matter (COM, 46 ± 3) and particulate organic matter (POM, 15 ± 1.4). Similar δ13C values were found for LMW‐DOM (−27.9 ± 0.5‰), COM (−27.4 ± 0.2‰), and POM (−26.2 ± 0.7‰), although there was a general increase with increasing size, suggesting a common terrigenous organic source. In contrast, distinct Δ14C values were found for LMW‐DOC (−155 to +91‰), COC (40 to 140‰), and POC (−467 to −253‰) with a decreasing trend from snowmelt to ice‐open season, suggesting that turnover pathways and transport mechanisms vary with organic matter size fractions. The high abundance of COC and its contemporary 14C ages points to a predominant source from modern terrestrial primary production, likely from the leaching/decomposition of fresh plant litter in the upper soil horizon. The predominately old POC (average 3698 ± 902 years B.P.), in contrast, was largely derived from riverbank erosion and melting of permafrost. These results imply that ice‐opening Yukon River flows are dominated by snowmelt (low δ18O) with high DOC (high Δ14C) but low DIC and Si(OH)4 concentrations, whereas late summer flows contain more products of permafrost or ice melt and rain (high δ18O), with low DOC (low Δ14C) but high DIC and Si(OH)4 concentrations. A warming climate with a deeper permafrost active layer in the Yukon River watershed would enhance the mobilization and export of old terrestrial OC, but largely in the particulate form into the Bering Sea and Arctic Ocean.