Insertion/deletion polymorphisms (Indels) have been considered as potential markers for forensic DNA analysis. However, the discrimination power of Indels is relatively lower due to the poor polymorphisms of diallelic markers. Here, two to three Indel loci that were very tightly linked in physical position were combined into a specific multi-Indel marker to improve the discrimination, as well as a multiplex that consisted of a set of multi-Indel markers was developed for forensic purpose. Finally, a multiplex system with 20 multi-Indel markers including 43 Indel loci from dbSNP database was constructed and DNA sample can be analyzed by this multiplex in one PCR reaction and one CE run. A total of 150 unrelated individuals from Hunan province in South-central China were genotyped by the multiplex system. The result showed that a total of 63 specific amplicons were detected, three alleles were observed in multi-Indel markers including two Indel loci and four alleles were observed in the markers including three Indel loci. The cumulative probability of exclusion and the accumulated discrimination power were 0.9989 and 0.9999999999994, respectively. Our result demonstrated that the strategy could be efficient to develop higher polymorphic multi-Indel markers, and the new multiplex could provide Supporting Information for forensic application.