Paeonia peregrina Mill. and Paeonia tenuifolia L. are endangered species of herbaceous plants with high ornamental and medicinal values. There are a few natural habitats in Serbia where these plants spontaneously grow: Krivi Vir, Skrobnica, Golina, Pirot, Deliblato Sands, and Sokobanja, in the municipality of Knjaževac. In July 2021, the symptom of leaf spots was observed on P. peregrina , while spots on leaves, stems, and lower branches on P. tenuifolia in the localities of Pirot and Deliblato Sands. Disease incidence was estimated to 32% in Pirot and 25-35% in Deliblato Sands. Therefore, the aim of the study was to identify the causal agent of leaf spot on P. peregrina and P. tenuifolia in Serbia by molecular identification and characterization, and morphological characterization. For molecular identification and characterization, nuclear ribosomal internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha ( EF-1α ), beta-tubulin ( TUB2 ) and histone 3 ( H3 ) genes were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, T1/Bt2b, and H3-1a/H3-1b, respectively. Morphological characterization of the representative isolates was done on potato dextrose agar (PDA) and potato carrot agar (PCA) media at 22 ◦ C under an 8 h light:16 h dark regime. Based on the molecular and morphological characteristics of the obtained isolates, Alternaria alternata was identified as the causal agent of leaf spot on P. peregrina and P. tenuifolia . In Serbia, to the best of our knowledge, this is the first report of A. alternata causing a leaf spot of P. tenuifolia .