The aim of this study was to compare the results of mycological and mycotoxicological analysis of two types of Slovak wheat samples, focusing on Alternaria genus. A total of 21 samples of conventional wheat grains and 3 samples of the new crossbred wheat were investigated for exogenous and endogenous mycobiota. The exogenous mycobiota was determined by using plate dilution method and the endogenous mycobiota by the method of direct placing of superficially sterilized grains on agar plates. Toxinogenity of selected isolates was analysed by means of thin layer chromatography. The obtained results of this study show a high isolation frequency of Alternaria isolates in samples of conventional as well as new crossbred wheat. A total of 4 species-groups of the genus Alternaria were isolated from conventional wheat (A. alternata, A. arborescens, A. infectoria, A. tenuissima) and 3 species-groups from new crossbred wheat (A. arborescens, A. infectoria, A. tenuissima). A. tenuissima species-group was isolated within the endogenous mycobiota from all samples of conventional and new crossbred wheat. Species-group with the second highest isolation frequency in all tested samples was A. infectoria. The highest relative density in all samples belongs to A. infectoria and A. tenuissima species-groups. Selected strains isolated from both types of wheat were tested for production of altenuene, alternariol monomethylether and alternariol. In neither case of A. infectoria species-group isolates was confirmed the production of tested mycotoxins. The highest toxinogenity (100%) was observed in strains of A. arborescens and A. tenuissima.