Abstract: 【Objective】Genetic diversity of sweet potato germplasm was analyzed based on chloroplast genome DNA spacer sequences(trnL-trnF, trnH-psbA and trnT-trnL), to provide theoretical basis for the protection, development and utilization of sweet potato germplasm.【Method】Taking 52 sweet potato materials from 12 different regions in China as the research object. From 10 primers of cpDNA spacer sequence, single, clear, bright and stable sequence primers were selected, and the selected spacer sequences were amplified by PCR, and sequenced and sequence spliced. The sequence features were analyzed by DnaSP 5.0 software. Using MEGA X software to calculate the genetic distance of 52 sweet potato germplasms and build phylogenetic tree.【Result】Seven pairs of primers with ideal amplification results were screened. The PCR amplified products were sequenced and analyzed, and a total of three effective markers(trnL-trnF, trnH-psbA and trnT-trnL) were obtained. The splicing sequence length of the three was 2239 bp, which contained 7 mutation sites, 2 singleton variable sites, 5 parsimony information sites, and 11 insertion/deletion sites. Among the 52 sweet potato materials, the number of variable sites(Vs) of trnL-trnF, trnH-psbA, and trnT-trnL sequences were 1, 1 and 5, respectively. The number of haplotypes(H) for the three were 2, 4 and 5, respectively. The number of haplotypes were 10 after three sequences being merged. The sequences with the highest nucleotide diversity(π) and haplotype diversity(H_dπ) were trnT-trnL(π=0.00052) and trnH-psbA(H_d=0.535). Tajima's D and Fu and Li's D^*, Fu and Li's F^* values of trnL-trnF, trnH-psbA and trnT-trnL sequences did not reached the level of significant difference(P>0.05), respectively, which indicated that variation of those chloroplast regions followed neutral theory of molecular evolution. Phylogenetic tree constructed based on combination sequences showed that genetic distance of 52 sweet potato varieties was 0-1.1848, and the average genetic distance was 0.1018. The combination sequences divided the test sweet potato varieties into 5 categories.There are only a small amount of germplasms in Type I to Type IV, and the remaining 41 germplasms were classified as Type V.【Conclusion】The genetic variation of 52 sweet potato germplasm materials is relatively rich, but the genetic diversity among germplasm materials is low, which is related to the cpDNA characteristics and the narrow genetic background of sweet potato. The trnL-trnF, trnH-psbA and trnT-trnL combination sequences can be used for sweet potato genetic diversity analysis, and the combination of the three sequences can distinguish the test materials into different groups and provide candidate materials for the next group breeding.