| Abstract: Objective To isolate, purify and identify the pathogenic fungi of black spot disease of Fritillaria thunbergii, and provide experimental basis for the monitoring and control of black spot disease of F. thunbergii. Methods Pathogenic tissue isolation method was used to isolate and purify pathogenic fungi from F. thunbergii. Partial fragments of ribosomal internal transcription spacer (ITS), translation elongation factor-1α (TEF-1α), glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and the second largest subunit of the nuclear RNA polymerase enzyme II (RPB2) of the fungi were obtained by PCR amplification with specific primers. After two-way sequencing and splicing, the amplified fragments were homologous with the corresponding sequences in GenBank, and the phylogenetic adjacent trees were constructed to identify fungal species. The pathogenicity of fungi to F. thunbergii was detected by friction inoculation method. Results Two fungi belonging to Alternaria genus were isolated and purified from F. thunbergii plants infected with black spot disease. The ITS, TEF-1α, RPB2, and G3PDH amplified fragments of the isolate 1 were 100% homologous to Alternaria alternata (accession numbers: MG182428, KY175227, XM_018523704, MG250634, respectively) in GenBank, while those of the isolate 2 was 100% homologous to A. tenuissima (accession numbers: MK967997, LC136862, KY290574, LT707523, respectively). Both of the fungi were identified as pathogenic fungi of F. thunbergii black spot by friction inoculation. Conclusion A. alternata and A. tenuissima are the pathogenic fungi of black spot disease of F. thunbergii. A. tenuissima was first reported to cause black spot of F. thunbergii.