A field survey was conducted during the barley growing season 2019 in the Karbala Province of Iraq. Barley plants with severe leaf spot symptoms were collected from several barley fields. The associated fungus was isolated and purified from those symptomatic leaves. The fungus colonies were aeriform mycelia in grey to black color. Conidia observed that they were light to dark brown and formed different-length chains. The causal agent was confirmed as Altenaria alternata via the PCR amplification and sequencing of the rDNA-ITS region, actA, and tef1 genes. Pathogenicity test showed that A. alternata isolated was pathogenic by producing light to dark brown spots on barley foliage. This is the first record of this pathogen causing Alternaria spot blotch on barley crops in Iraq. Keywords: Alternaria alternata; leaf spot; Hordeum vulgare; Pathogenicity assessment; Phylogeny analysis.