Abstract: Objective To conduct genotyping and molecular tracing analysis on Plasmodium vivax samples from a cluster of P. vivax malaria outbreak in order to provide a reference for case geographical origin determination. Methods Blood samples from 4 patients in a vivax malaria cluster in Longhui County, Hunan Province from June to July 2018 were collected for species identification by qPCR, and 9 microsatellite molecular markers were used to genotype the parasite strains from four samples. The population genetic STRUCTURE analysis was performed based on the VivaxGEN-MS microsatellite genotype database of P. vivax in the Asia Pacific Malaria Elimination Network, to determine the genetic subgroups and geographical origin of the strains. Results By qPCR, all 4 cases were identified as Plasmodium vivax infection, and 9 microsatellite loci of the 4 cases were successfully typed, and the four samples had different genetic haplotypes, among which case 1, case 3, and case 4 were infected by a single clonal strain, and case 2 was infected by a polyclonal strain. When all P. vivax samples were divided into 2 subpopulations (K=2) by STURCTURE analysis, 4 Hunan samples were classified into tropical genetic subpopulations (comprising strains from Ethiopia, Iran, Bhutan, Malaysia, Indonesia, and southern China). When the samples were divided into 4 subgroups by STURCTURE analysis (K=4), the 4 Hunan samples were classified as South Asian/Southeast Asian genetic subgroups (originating from Bhutan, Malaysia, Indonesia, and southern China). Conclusions The results of molecular tracing do not support that the 4 P. vivax strains in this outbreak originated from the population of central China. The technology of molecular tracing of P. vivax can provide objective evidence for determining the source of infection in malaria cases during the stage of malaria elimination and post-elimination.