This study aimed to develop a multiplex-PCR for quick and reliable detection of Chlamydia trachomatis infection. The optimal conditions of PCR reactions to detect specific DNA fragment of Chlamydia trachomatis with 4 primer pairs CtP, CtM, CtR, NO, Chlamp, and positive control primers, AR, were investigated. The results showed that the optimal annealing temperature of each primer-pairs ranged from 53°C to 57°C. Besides, the optimal annealing temperature of the multiplex PCR reaction which includes AR, NO, CtP and Chlamp primers was identified as 56°C. The rate of infection in gynecological patients in Bac Giang Hospital was 12.35 percent, as there are 31 positive samples in a total of 251 samples.