This study investigates the link between oligonucleotide (ON) structure and diastereomer separation in hydrophilic interaction chromatography (HILIC). The authors identify higher-order structures (HOS) as the primary cause of diastereomer separation in HILIC. Using conformational predictions, melting profiles, mass spectrometry, and HILIC analysis, they demonstrate that intrinsic HOS play a crucial role in partial diastereomer separation. The study also shows how various chromatographic parameters, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier, can be adjusted to enhance or suppress diastereomer separation. This work significantly facilitates the characterization of therapeutic ONs, particularly those with phosphorothioate (PS) modifications, by enabling the monitoring of batch-to-batch diastereomer distributions in full-length siRNAs. The findings highlight the importance of understanding ON HOS and their interactions with the stationary phase and mobile phase to optimize HILIC-based analysis.This study investigates the link between oligonucleotide (ON) structure and diastereomer separation in hydrophilic interaction chromatography (HILIC). The authors identify higher-order structures (HOS) as the primary cause of diastereomer separation in HILIC. Using conformational predictions, melting profiles, mass spectrometry, and HILIC analysis, they demonstrate that intrinsic HOS play a crucial role in partial diastereomer separation. The study also shows how various chromatographic parameters, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier, can be adjusted to enhance or suppress diastereomer separation. This work significantly facilitates the characterization of therapeutic ONs, particularly those with phosphorothioate (PS) modifications, by enabling the monitoring of batch-to-batch diastereomer distributions in full-length siRNAs. The findings highlight the importance of understanding ON HOS and their interactions with the stationary phase and mobile phase to optimize HILIC-based analysis.