Dendrocalamus brandisii is a high-quality bamboo species that can be used for both bamboo shoots and wood. The nutritional components and flavors of D. brandisii vary from different geographical provenances. However, the unique biological characteristics of bamboo make morphological classification methods unsuitable for distinguishing them. Although the new cultivar 'Manxie No.1' has significant differences in the branch characteristics and the color of shoot sheaths compared to the D. brandisii, it still lacks precise genetic information at the molecular level. This study identified 231,789 microsatellite markers based on the whole genome of D. brandisii and analyzed their type composition and distribution on chromosomes in detail. Then, using TP-M13-SSR fluorescence-labeling technology, 34 pairs of polymorphic primers were screened to identify the new cultivar 'Manxie No.1' and 11 different geographical provenances of D. brandisii. We also constructed DNA fingerprinting profiles for them. At the same time, we mapped six polymorphic SSRs to the gene of D. brandisii, among which SSR673 was mapped to DhB10G011540, which is related to plant immunity. The specific markers selected in this study can rapidly identify the provenances and the new cultivar of D. brandisii and help explore candidate genes related to some important traits.