We investigated the polymorphism of five X-chromosomal short tandem repeat markers (ChrX STRs) loci (DXS7132, DXS981, DXS6803, DXS6809, and DXS6789) and their value for forensic applications. A fluorescent multiplex polymerase chain reaction (PCR) for amplifying five ChrX STRs loci in the same PCR reaction was set up. A total of 827 unrelated individuals of the Han nationality in China were tested. The results show that the five loci in the multiplex system provide high polymorphism information for forensic identification and paternity testing, particularly for difficult paternity-deficient cases.