Severe leaf spot disease was observed on Aloe vera plants in the winters of 2011 and 2012 during a survey of various nurseries of Gwalior, India. Irregular, sunken, dark creamish brown spots having reddish brown margin were noticed on both surfaces of the leaves. The causal organism was consistently isolated from symptomatic leaves on potato dextrose agar media (PDA). A total 59 isolates of fungi were recovered from diseased A. vera leaves, and 37 isolates were identified as belonging to the genus Fusarium. On the basis of morphological characteristics and internal transcribed spacer (ITS) region of rDNA amplified using the primers ITS4/ITS5 the pathogen was identified as Fusarium proliferatum (Matsushima) Nirenberg and pathogenicity of the isolate was confirmed by using Koch’s postulates. To the best of our knowledge, this is the first report of leaf spot disease caused by Fusarium proliferatum on A. vera plants in India.