Non-ionic surfactants consist of hydrophobic alkyl chains and hydrophilic polyethylene oxide (EO), whose distributions significantly influence properties such as hydrophilic-lipophilic balance and critical micelle concentration. In particular, secondary alcohol ethoxylates (AEOs), in which EO units are inserted within the alkyl chain, are complex mixtures that include multiple positional isomers in addition to homologous EO and alkyl‑chain distributions, making them difficult to separate via conventional liquid chromatography (LC) or gas chromatography (GC). Therefore, we evaluated supercritical fluid chromatography (SFC) for high-resolution analysis of these nonvolatile mixtures. Using an octadecylsilane (ODS) column without end‑capping (Inertsil ODS‑4 noEC, 2.1 mm inner diameter (i.d.) × 150 mm, 3 μm) connected in series (five columns, total length 750 mm), with a flow rate of 0.8 mL/min, column oven at 40 °C, and back pressure regulator set at 10 MPa, we achieved separation of positional isomers of secondary AEOs that had been previously unresolved. For example, five positional isomers of the C14 alkyl chain with EO6 were resolved (five peaks) in a single analysis (total run time 200 min), and the measured peak capacity was 67. This separation was due to differences in the molecular shapes of the positional isomers of secondary AEOs, which resulted in varying degrees of steric hindrance from the ODS group on the stationary phase surface and differences in polar interactions with the residual silanol groups. SFC is useful for separation and composition analysis of complex mixtures that are difficult to separate via GC or LC and is expected to be utilized for the precise analysis of components such as surfactants and synthetic polymers whose composition significantly affects performance and quality.