The Ob-Irtysh River system is the seventh-longest one in the world. Unlike the other Great Siberian rivers, it is only slightly impacted by the continuous permafrost in its low flow. Instead, it drains the Great Vasyugan mire, which is the world largest swamp, and receives huge load of the Irtysh waters which drain the populated lowlands of the East Siberian Plain. The central challenge of this paper is to understand the processes responsible for molecular transformations of natural organic matter (NOM) in the Ob-Irtysh river system along the South-North transect. For solving this task, the NOM was isolated from the water samples collected along the 3,000 km transect using solid-phase extraction. The NOM samples were further analyzed using high resolution mass spectrometry and optical spectroscopy. The obtained results have shown a distinct trend both in molecular composition and diversity of the NOM along the South-North transect: the largest diversity was observed in the Southern “swamp-wetland” stations. The samples were dominated with humic and lignin-like components, and enriched with aminosugars. After the Irtysh confluence, the molecular nature of NOM has changed drastically: it became much more oxidized and enriched with heterocyclic N-containing compounds. These molecular features are very different from the aliphatics-rich permafrost NOM. They witnesses much more conservative nature of the NOM discharged into the Arctic by the Ob-Irtysh river system. In general, drastic reduction in molecular diversity was observed in the northern stations located in the lower Ob flow.