The Primula chloroplast genomes exhibited moderate sequence divergence. Furthermore, the results showed that the sequence of coding regions and the two IR regions were significantly more conserved than that of LSC and SSC regions. In the coding regions, most genes were relatively conserved, except for matK , rpl22 , ndhFand ycf1 . In contrast, the intergenic regions were shown to be highly divergent (Figure 3). Then, we found that the variation level of DNA polymorphism was from 0.00067 to 0.02633. The greatest differences among the five Primula species were located in the two SC regions, while IR regions were the least different. About 17 hyper-variable regions were discovered with a nucleotide diversity (P_i ) value that was greater than 0.01 (Figure 4). Some relatively high P_i value were detected in CDS, such as psbA (0.02633), ycf1 (0.01533), psaJ(0.01333), rpl32 (0.01233), petL (0.01167) and ndhA(0.01033). Consistently, the gene ycf1 exhibited higher diversity and showed abundant variation. In addition, we revealed five highly divergent regions among noncoding regions, including ccsA-ndhD(0.01733), ndhF-rpl32 (0.01583), petA-psbJ (0.01567),trnE (UUC)-trnT (GGU) (0.01267) and rps15-ycf1(0.01167). These mutational hotspots can serve as potential loci when developing novel DNA barcodes for plant classification.