In 2020, a leaf spot disease of hemp (Cannabis sativa) was found in Wuwei, Gansu, China. In order to determine the causal agents, a total of 29 Alternaria-like strains were collected. Based on morphology and phylogenetic analysis, these strains were identified as two members of Alternaria alternata Species Complex (AALSC), one monotypic lineage of A. helianthiinficiens and a new species of Alternaria cannabina in section Infectoriae. The new species was identified and illustrated here based on morphology and sequence analyses of the internal transcribed spacer rDNA regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), translation elongation factor (tef1), RNA polymerase II subunit (rpb2) and plasma membrane ATPase (ATPase) genes. Moreover, pathogenicity tests of all four taxa showed that strains of AALSC were more aggressive to hemp leaves than the other two species.