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The Karst landform is the main geographic characteristic in South China. Such areas are rich in vegetation and especially suitable for growth of shrubs and herbaceous plants. In this study, 11 <i>Septoria</i> strains were obtained from different plants’ leaves collected in the Kunming Botanical Garden, Yunnan Province, China. Based on single-gene and multi-gene analyses of five gene loci (<i>tef1</i>, <i>rpb2</i>, <i>tub2</i>, ITS, and <i>LSU</i>) and four gene regions (without <i>LSU</i>), these strains were found to belong to three independent phylogenetic lineages representing five species, including four novel taxa, and one new record for China. Five single gene trees were also provided to evaluate the effectiveness of each gene for discriminating the species, as a result of which <i>tub2</i> was found to have the most suitable DNA barcode for rapid identification. Morphological descriptions, illustrations, and comparisons are provided for a more comprehensive assessment. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) with a pairwise homoplasy index (PHI) test was used to evaluate the conclusions of the phylogenetic analyses.