The genus Fritillaria is an extremely complicated group in phylogeny and taxonomy, which contained numerous medicinal species. Both traditional characteristic-based taxonomy and standard DNA barcodes (ITS, trnH-psbA and rbcL) are difficult to effectively delimit its species boundaries. Here, we generated a large dataset of chloroplast genomes from multiple accessions per species of Fritillaria to test and evaluate their effectiveness in species discrimination. Moreover, phylogenetic inference and divergence time estimation were performed based on the new sequences and published chloroplast genomes. The results showed that only F. cirrhosa could not be correctly discriminated in all species by using complete plastomes, which suggested that they could enhance species discriminatory resolution, but was not omnipotent. Furthermore, four regions (ycf1, matK-trnG-GCC, rpoC1 and matK) gained remarkably higher resolution than that of other plastid regions, but only matK might be suitable to identify Fritillaria species in consideration of their lengths. Phylogenetic inference showed that the subgenus Fritillaria of China was divided into four major clades, and taxonomic delimitation of F. omeiensis and F. hupehensis in Flora of China (2000) might be questionable and needed further revision. Molecular dating demonstrated that both origin and divergence of subgenus Fritillaria, as well as its four major clades, were significantly associated with geological and climatic fluctuations during the Middle to Late Miocene. This study would enrich case studies of super-barcoding and provide new insights on speciation, lineage diversification and biogeography of the genus Fritillaria.