Alternariosis is one of the most common tomato diseases that cause plant development retardation, yield decrement and fruit quality deterioration. We identified Alternaria infection in tomato plants during the whole vegetation cycle in absence of disease’s symptoms via molecular methods. The main goal of this study was to quantify of Alternaria spp. in tomato varieties of IGFPP selection that differ in susceptibility to some fungal infection using nested-PCR assay. Were studied plants of three tomato varieties – ‘Cerasus’, ‘Exclusiv’ and ‘Elvira’. Congregate leaf samples were collected from 12 asymptomatic plants of each variety at every phase of plant vegetation. DNA was extracted from 1g of plant material following CTAB extraction protocol. A set of home-designed primers for Alternaria spp. detection was created on the basis of RNA polymerase II second largest subunit (RPB2) gene sequences from NCBI nucleotide collection. The PCR was performed in a 25μl mix containing DreamTaq buffer and 1,2 U DreamTaq DNA polymerase, 0,2 mM of each dNTPs, 5 pM of each primer and 50 ng of DNA. The first round of nested-PCR included 1 cycle at 95°C for 3 min, followed by 30 cycles: 950C –1 min, 600C – 1 min, 720C – 1 min. The second round – the same conditions except initial 1 cycle at 95°C for 3 min. The results of the molecular analysis demonstrated the presence of Alternaria spp. in all DNA samples extracted from tomato plants during vegetative season (year 2019). For semiquantitative analysis were selected DNA samples obtained at the point of mass manifestation of alternariosis’ symptoms. Equivalent quantity of total DNA of each variety was used in series of sequential five-fold dilutions and applied in nested-PCR. The absence of a positive signal in the sample means that there is no pathogen DNA in diluted partition. Based on the results of the molecular analysis, given the dilution index, the number of pathogen’s DNA copies contained in the initial sample was calculated. So, the comparative content of Alternaria DNA in the studied varieties as follows: in variety ‘Cerasus’ it is 5 times lower than in ‘Elvira’ and 25 times compared to ‘Exclusiv’. Therefore, ‘Cerasus’ variety showed the lowest susceptibility to Alternaria spp., and ‘Exclusiv’ – the highest.