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Fungi were isolated from diseased maize samples suffering from stalk rot. Six isolates were identified as Fusarium thapsinum on morphological and microscopic features. These were further verified by PCR analysis with EF-1α gene amplification. The phylogenetic analysis demonstrated that all the isolates of F. thapsinum were more than 98% similar based on the maximum likelihood tree analysis and were grouped into a single major cluster. Pathogenicity test was conducted when maize achieved its physiological maturity stage. Pathogenicity assay demonstrated the ability of all the six isolates to cause infection. Results showed that disease severity index was ranged from 39–47%, reflecting variation in aggressiveness among the isolates. Isolates MN207144, MN200604, MN200605, and MN200606 were found highly aggressive. In the light of previously available documented evidences, it is the first report of F. thapsinum associated with stalk rot of maize in Punjab, Pakistan.