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[Display omitted] • Paleoecological and paleohydrological changes in the Qaidam Basin occurred during the EOT. • Changes in CPI, ACL, Paq and L/H, indicating bacterial, algal and aquatic plants shrink , and terrestrial plants flourish. • δ2H 31 values were increased by 18.1‰, indicating a decrease in water vapor content and an increasingly arid climate. The Eocene/Oligocene transition (EOT) at ∼34 Ma has been considered the most prominent climatic cooling event in the Cenozoic Era. However, few organic biomarker studies have attempted to reconstruct relevant terrestrial ecological and hydrological responses to an age characterized by drastic temperature changes. This research aims to reconstruct the paleoecological and paleohydrological evolution of the Qaidam Basin (QB) on the northeastern Tibetan Plateau (NE TP) between 40.2 and 29.9 Ma, based on analyses of n -alkane chain length distributions as well as compound-specific hydrogen isotopic compositions (δ2H alk). Research showed the presence of a sequence of pronounced ecological and hydrological variations at 35.0–33.1 Ma, including: 1) a significantly decreased input of bacteria, algae and aquatic plants, allied with a markedly increased input of terrestrial plants, reflected in a significant increase in carbon preference index (CPI) and the average chain length (ACL) ratio, as well as an abrupt decline in the relative ratio of mid- to long-chain length homologue (Paq) values and the ratio of low- to high- molecular-weight n -alkanes (L/H); and 2) drier climatic conditions, reflected in an 18.1‰ increase in δ2H 31 values, as well as more positive hydrogen isotopic precipitation values (δ2H p). These features demonstrate significant paleoecological and paleohydrological changes in the QB in response to the drier and cooler climate during the EOT. [ABSTRACT FROM AUTHOR]