The chromosome microdissection, cloning and painting technology has evolved into an efficient tool for genomic research. Application of these techniques has rarely been applied for forest plants, largely due to the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection, cloning and painting in forest plants using poplar (Populus tremula) as a model. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by the Sau3A linker adaptor mediated PCR (LA-PCR) technique, by which 200bp to 3,000bp smear DNA fragments were obtained. Then, the second round PCR products from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. Approximately 3 x 105 recombinant clones were obtained. The second round PCR products were used as a complex probe mixture for fluorescent in situ hybridization (FISH) on the metaphase spreads of poplar. Hybridization signals were observed, mainly, along the entire chromosome 1, at the same time, signals were also present on telomeric and centromeric regions of other chromosomes. Therefore, this research suggests that chromosome microdissection, cloning and painting of the single small chromosome in forest plants are feasible.