Bamboo plants have a distinctive life cycle with long flowering periodicity. Many species remain in vegetative growth for decades, followed by large-scale flowering and subsequent death. Floral transition is activated while shoot buds are still dormant in bamboo plants. In this study, we performed morphological characterization and transcriptome analysis of the shoot buds at different growth stages from flowering and non-flowering Pleioblastus pygmaeus. The morphological and anatomical structures of the dormant shoot buds were similar in flowering and non-flowering plants, while there was an obvious difference between the flower buds from flowering plants and the leaf buds from non-flowering plants. The transcriptomes of the dormant shoot buds, germinated shoots, and flower buds from flowering P. pygmaeus, and the dormant shoot buds, germinated shoots, and leaf buds from non-flowering P. pygmaeus were profiled and compared by RNA-Seq. The identified sequences were mostly related to metabolic synthesis, signal transmission, translation, and other functions. A total of 2434 unigenes involved in different flowering pathways were screened from transcriptome comparisons. The differentially expressed unigenes associated with the photoperiod pathway were related to circadian rhythm and plant hormone signal transduction. Moreover, the relative expression levels of a few key flowering-related genes such as CO, FT, FLC, and SOC1 were quantified by qRT-PCR, which was in accordance with RNA-Seq. The study revealed morphological differences in the shoot buds at different growth stages and screened flowering-related genes by transcriptome comparisons of the shoot buds from flowering and non-flowering P. pygmaeus, which will enrich the research on reproductive biology of bamboo plants and shed light on the molecular mechanism of the floral transition in bamboo plants.