Lonicera japonica Thunb. (honeysuckle) is widely cultivated as a medicinal herb in China. In recent years, the crop has been affected by a leaf spot disease in Suiyang, Guizhou, China. Disease symptoms included necrotic spots and wilting on leaves. The pathogen responsible for the disease was successfully isolated from symptomatic leaf tissue and identified. Identification based on morphological characteristics suggested that all the recovered fungal isolates belonged to small-spored Alternaria species. Multilocus sequence analyses of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), endopolygalacturonase (endoPG), translation elongation factor 1-alpha (tef1), and Alternaria major allergen (Alt a 1) genes, assigned five selected isolates as belonging to Alternaria alternata. Pathogenicity assays confirmed the Alternaria alternata species caused the observed symptoms. To our knowledge, this is the first report of honeysuckle leaf spot caused by A. alternata in China. The results of this study advance our understanding of honeysuckle leaf spot in China and lay the groundwork for developing efficient pre- and post-harvest control measures to stop the spread of the disease.