Single enantiomers of three 1,2-substituted ferrocene derivatives, i.e. 1-methoxymethyl-2-hydroxymethylferrocene (1), 1-formyl-2-hydroxymethylferrocene (2) and 1-iodo-2-hydroxymethylferrocene (3), sharing the common hydroxymethyl substituent and the presence of planar chirality only, were investigated for their spectroscopic (IR and UV) and chiroptical (VCD and ECD) properties. Both enantiomers of 1 were obtained for the first time in optically pure form by lipase-catalyzed kinetic resolution of the corresponding racemate (±)-1 and separately converted into formyl derivatives (+)-2 and (–)-2.The experimental spectroscopic and chiroptical data were compared with DFT calculated spectra and excellent correspondence was found for all compounds, allowing one to confirm the previously assigned absolute configurations. The common features in the VCD spectra of a doublet between 940 and 965 cm−1 and the short-wavelength (about 200 nm) doublet and the longest wavelength band in the ECD spectra were analyzed to test whether they may be taken as markers of the absolute configuration (AC). The predominance of conformers with intramolecular hydrogen bond for the first two investigated compounds is predicted by conformational analysis and also confirmed by NMR.