Being organelles present in higher plants and algae, and containing the entire machinery for the process of photosynthesis and their own genetic system, chloroplasts have played important roles in plant evolution. With the help of molecular approaches, the structure and sequences of chloroplast genomes have been studied to a large extent. The valuable chloroplast genomic information has been utilized in investigating plant evolution, diversity and phylogenetic relationships between different species. At the same time, chloroplast genome sequences are the essential information paving the road for successful chloroplast genetic engineering, which has shown considerable advantages compared with nuclear transformation and demonstrated its great potential in genetic improvement and the production of biopharmaceuticals. The latest developments in chloroplast genome analysis are reviewed, providing readers with insight into the fast growing field and facilitating a comprehensive understanding of the evolution, genetics and phylogenetic relationships among plants. It is also hoped to promote the further development of chloroplast technology and the commercial application of chloroplast transformation.