Ustilaginoidea virens, the false smut pathogen of rice, produces false smut balls on spikelets after heading. To clarify how the fungus invades spikelets during the booting stage, we developed a fungal strain that expresses a green fluorescent protein gene and injected conidia from this strain into rice sheaths. Observations at 48 h post-inoculation showed many conidia were present on spikelet surfaces, and the conidia had germinated and the hyphae have gradually grown by 120 h post-inoculation. By 144 h, hyphae had invaded spikelets through their apices, via the small gap between the lemma and palea and had already reached all floral organs.