Reversed-phase high-performance liquid chromatography (RP-HPLC) was used for the separation of 3-hexylthiophene oligomers in the range of 3 to 30 monomeric units, while systematically varying stationary and mobile phases. A set of different columns was chosen, covering a broad range of silica types, pore sizes and bonding chemistry. Mobile phases of tetrahydrofuran (THF) combined with water, acetonitrile (ACN) or methanol (MeOH) were used. Although differences between columns were small, a higher selectivity correlated with a lower hydrophobicity parameter from the Galushko column test. The model of Jandera, based on the linear solvent strength model of Snyder, was used to describe the retention of the oligomers in gradient mode. This gave information about selectivities on different stationary phases similar to the hydrophobicity parameter. Contrary to the stationary phase, the mobile phase had a major influence on the selectivity. The THF–water combination gave much higher selectivities compared to THF combined with MeOH or ACN. Using the aqueous mobile phase even enabled separation of different isomers. Determination of thermodynamic parameters for the model compounds showed that retention of the different isomers was mainly determined by the orientation of the side chains at both ends of the chain. An additional repeating unit in the middle of the polymer backbone gave a similar contribution to retention, irrespective of the orientation of its side chain. Three model isomers were separated by preparative RP-HPLC and identified by proton nuclear magnetic resonance spectroscopy. The combination of subsequent preparative size-exclusion chromatography, RP-HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry enabled the identification of the two major oligomeric series in the sample as the regioregular product with one bromine end group and, in smaller amounts, a regioirregular product with two bromine end groups.