To identify Chlamydia trachomatis (CT) endocervical infection among gestations at risk for preterm birth by means of DNA amplification. One hundred fifty eight (n = 158) gestations of Guayaquil (63.3%) and Quito (36.7%) Ecuador, at risk for preterm birth: threatened preterm labor (TPL, 60.8%) and preterm premature rupture of membranes (PPROM, 39.2%) were recruited. CT infection was presented in 8.2% of cases (TPL: 10.4% and PPROM: 4.8%). No significant difference was observed in this frequency in regard to city or diagnosis. Although the causative role of CT infection for these antenatal complications cannot be completely ruled out, its presence may justify an antenatal routine screening program in order to provide appropriate therapy.