Boll rots of cotton (Gossypium hirsutum L.) are common in the humid areas of the Southeastern US. One type of boll damage that may be differentiated from others is hardlock, with symptoms that include compression of the fibers within individual locules of mature, open cotton bolls without further obvious disintegration of the lint or damage to the carpel wall. The principal economic effect is that the boll's lint is unharvestable by mechanical cotton pickers. This disease is endemic to the Southeast and can cause severe yield losses up to 70% in some fields. Scanning electron microscopy images of fibers from hardlocked bolls showed flattened and twisted tissue compared to fibers from healthy bolls. Fusarium verticillioides (Saccardo) Nirenberg was the fungus most commonly isolated from seeds of developing cotton bolls. Flowers inoculated with F. verticillioides on the day of bloom by spraying a spore suspension onto the flowers developed significantly (P < 0.05) more hardlock symptoms compared to untreated controls. The infection process was analyzed using a F. verticillioides isolate tagged with green fluorescent protein (GFP). When it was applied to cotton flowers on the day of bloom, the GFP-tagged F. verticillioides strain was detected in the stigma and style by 2 days after bloom (DAB) and in developing seeds at 4, 6, 8, 10, 16, 20, 40, and 60 (open bolls) DAB. By 8 DAB, the GFP F. verticillioides was isolated from over 80% of developing seeds.