The life cycle of the sugarcane smut fungus Sporisorium scitamineum is a multistep process. Haploid sporidia of compatible (MAT-1 versus MAT-2) mating types fuse to generate pathogenic dikaryotic hyphae to infect the host. Within the host tissues, diploid teliospores are formed and induce a characteristic sorus that looks like a black whip. The diploid teliospores germinate to form haploid sporidia by meiosis. In order to monitor fungal development throughout the whole life cycle, we expressed the green fluorescent protein (GFP) and red fluorescent protein (RFP) in S. scitamineum MAT-1 and MAT-2 sporidia, respectively. Observation by epifluorescence microscope showed that conjugation tube formation and sporidia fusion occurred at 4 to 8 h, and formation of dikaryotic filaments was detected at 12 h after mating. The resultant teliospores, with diffused GFP and RFP, underwent meiosis as demonstrated by septated hypha with single fluorescent signal. We demonstrated that GFP- and RFP-tagged strains can be used to study the life cycle development of the fungal pathogen S. scitamineum, including the sexual mating and meiosis events. This dual-color imaging system would be a valuable tool for investigation of biotic and abiotic factors that might affect the fungal life cycle development and pathogenesis.