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The chromophoric dissolved organic matter (CDOM) is a complex mixture of organic biochemicals that can undergo various pathways of diagenetic alteration. The CDOM optical properties are applied in aquatic ecosystems monitoring and remote sensing. Fluorescence spectra of different CDOM types may vary in wavelength of emission maximum, fluorescence quantum yield values and their dependence upon excitation wavelength. Despite its role in the sustainability of life, the basic chemical nature of CDOM is still poorly understood. The process of diagenesis can create a chain of various hydrophobic/hydrophilic molecules as a result of oxidation/cleavage and condensation reactions. Thus, any effort to understand the ecological fate of these compounds in the environment requires identification of their hydrophobic properties. The objectives of the current work was to apply fluorescence spectroscopy and reversed-phase high performance liquid chromatography (RP HPLC) with on-line absorbance and fluorescence detections to analyze CDOM from three Karelian lakes (Onego lake, as well as from two small lakes of marsh origin at the Karelian site of the White Sea) to reveal the fluorescent constituents of different polarity. Fluorescence emission spectra were registered with luminescence spectrometer for different excitation wavelengths varying from 230 to 560 nm. The CDOM fluorescence quantum yield was determined using a solution of sulphate quinine as a reference. Fluorescence quantum yield and emission maximum wavelength were described as a function of excitation wavelength. So-called “blue shift” of emission was observed with change in λex from 270 to 310 nm; fluorescence quantum yield reached maximum at λex~370/380 nm and decreased monotonically thereafter. The RP HPLC with stepwise gradient formation using methanol and aqueous phosphate buffer gave possibility to map CDOM in terms of distribution of hydrophilic/hydrophobic chromophores. The chromatograms with on-line absorbance detection from 220 nm to 370 nm exhibited the resolution of seven peaks with different hydrophobic properties. The hydrophobicity of peaks increased from the first to the last eluted peak due to the increase of methanol concentration in the stepwise separation procedure. The highest content of hydrophilic fraction was found in the CDOM from the Onego lake. "Hydrophobicity profiles" (peaks area taken at rising detection wavelength) were similar for all lakes. In the wavelength range 220-270 nm hydrophilicity increased with maximum at 260-270 nm and corresponded to absorption bands of aromatic amino acids and simple phenols. Between absorbance wavelengths 270 and 370 nm the significant growth of hydrophobicity was found. The on-line fluorescence detection with λex =270 nm shown that CDOM in all three lakes contained fluorophores with humic-type fluorescence maximum at 420-450 nm, depending on hydrophilicity/hydrophobicity of the fraction. The most intensive humic-type fluorescence with maximum at 420 nm (Onego lake) or 435 nm (marsh lakes) was found for the hydrophilic peaks. Along with rising hydrophobicity of the fraction the humic-type fluorescence maximum shifted towards longer wavelengths. The fluorophores emitting protein-type fluorescence with maximum around 345 nm were found in the most hydrophobic fractions of CDOM from the Onego lake. The data obtained could be significant for future CDOM structural characterization and application in remote sensing. The work has been supported by Russian Foundation for Basic Research (project no 13-05-00241-a).