The industrially important bamboo, Dendrocalamus hamiltonii Nees et Arn. ex Munro is monocarpic and flowers gregariously only once after 40–60 years. The age of most of the plants growing in the wild is not known. This makes it impossible to distinguish between the juvenile and aged plants that are about-to-flower and die. Conservation of juvenile plants, but utilization of aged ones is therefore, extremely difficult. Since identification of genes specific to about-to-flower plants was envisaged to facilitate this differentiation between juvenile and aged plants, de novo transcriptome analysis of vegetative, about-to-flower and flowering in vitro shoots of D. hamiltonii was undertaken. A total of 195 differentially-expressed-genes were identified. Key genes specific to about-to-flower stage (AFS) were identified and validated by q-PCR in plants growing in wild. The q-PCR expression patterns of selected genes including the key regulators of floral transition i.e., CONSTANS-like (COL), phytochrome interacting factor 5 (PIF5), VERNALIZATION INSENSITIVE 3 (VIN3), flowering time control protein (FCA), transcription factor GAMYB (GAMYB), squamosa promoter-binding-like protein 3 (SPL3), heading date 3a (Hd3a), apetala 2 (AP2) and MADS box genes matched exactly in the about-to-flower shoots (AFS) of in vitro and sporadic about to flower shoots (SAFS) of bamboos growing in the wild. These can serve as markers of ‘floral transition’. Establishment of a close homology of D. hamiltonii with D. latiflorus Munro, Phyllostachys edulis (Carriere) J. Houzeau, Bambusa oldhamii Munro and B. multiplex (Lour.) Raeusch. ex Schult. through multiple sequence alignment and phylogenetic analysis of PIF5, VIN3, SPL3, and MADS-box transcription factor 15 (MADS15) suggested the potential applicability of identified genes in other industrially-important-bamboos. Transcriptional corepressor LEUNIG (LUG), transcription regulatory protein SWI3D (SW13D), protein HOTHEAD (HTH) and E3 ubiquitin ligase BIG BROTHER-related (BBR) were identified in D. hamiltonii for the first time. The q-PCR analysis of identified genes can serve as potential tool for distinguishing juvenile and about-to-flower aged bamboo plants growing in the wild. The study is a step towards the conservation of juvenile bamboos as a sustainable resource base for future industrial applications.