Sequencing of cloned PCR-amplified species-specific rDNA fragments and isolation of axenic cultures from tomato fruits was carried out to study the mycobiota of tomato leaves and fruits in European part of Russia. DNA was extracted from the leaves, and library of ITS region fragments was constructed in E. coli by cloning of PCR products. This survey revealed fourteen species associated with disease-affected leaves: Septoria lycopersici, Fulvia fulva (=Cladosporium fulvum), Didymella glomerata (=Phoma glomerata), Cladosporium herbarum, Podosphaera fusca, Neocamarosporium goegapense (=Phoma betae), Rhizoctonia solani, Candida albicans, Dioszegia hungarica, Cladosporium cladosporioides, Didymella lycopersici, Alternaria infectoria, Alternaria alternata, Cryptococcus tephrensis. In the leaves from healthy plants without any visible symptoms DNA of three species was found: Aspergillus versicolor, Alternaria alternata, Aureobasidium pullulans. Analysis of axenic cultures isolated from green diseased tomato fruits revealed fungal species: Alternaria alternata, Alternaria solani, Phomopsis phaseoli, Fusarium equiseti, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Colletotrichum coccodes. This research provides new information on the mycobiota of tomato in Southern Russia, the main tomato producing region of the country. © 2020, University of Zagreb - Faculty of Agriculture. All rights reserved.