Recent research on chloroplast genome focused on the sequencing of new plastid genomes and comparing related genomes. Influence exerted by non-adjacent sites is rarely mentioned. In the current study, the substitution sites were counted based on the pairwise comparison of five Poaceae chloroplast genomes, using Phalaenopsis aphrodite chloroplast genome as the outgroup. The relationship between mutation patterns and the base composition of flanking sites was detected. A significant flanking sites effect was observed. Substitutions to and from each dinucleotide were calculated, and three strong “losers” (AA, AT, and TA) and four strong “gainers” (CC, CG, GC, and GG) were found. The number of AA is higher in the ancestral sequence which gradually decreased in the evolution process. The reduction in A and T with C and G accumulation are reported as well. The dinucleotide substitution trends are largely determined by the mononucleotide substitution trends. Results indicate that context significantly influences mutations, further enhancing our understanding of context dependency and mutation dynamics in chloroplast genomes.