Onion (Allium cepa L.) is one of the most important vegetable crops in Serbia, where it is grown on an approximate surface of 20,000 ha. During the routine quality control analysis of onion seed in 2014, fungal infection was observed in an average of 28% of the seed. The objective of this paper was to isolate, determine, and identify Fusarium sp. based on the pathogen's morphological and molecular characteristics. Onion seed samples were collected from different localities in the region of Vojvodina. To obtain a DNA sequence-based identification, a total DNA of the 25 isolates was extracted directly from the mycelium (~ 100 mg wet weight), with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Following DNA extraction, the translation elongation factor 1-alpha region was amplified by PCR with the primer pair EF1 and EF2. An amplicon of 700 bp was amplified in 25 tested isolates. Identification of one isolate was performed by sequencing the translation elongation factor EF1α gene, which was deposited in the NCBI GenBank database under accession number KP658211 (Fusarium sp. FIESC3).