To compare the second-trimester plasma cell-free (PCF) transcriptome of women who delivered at term with that of women with spontaneous preterm birth (sPTB) at or before 32weeks of gestation and identify/validate PCF RNA markers present by 16weeks of gestation. Prospective case-control study. Academic tertiary care centre. Pregnant women with known outcomes prospectively sampled. PCF RNAs extracted from women at 22-24weeks of gestation (five sPTB up to 32weeks and five at term) were hybridised to gene expression arrays. Differentially regulated RNAs for sPTB up to 32weeks were initially selected based on P value compared with control (P<0.01) and fold change (≥1.5×). Potential markers were then reordered by narrowness of distribution. Final marker selection was made by searching the Metacore™ database to determine whether the PCF RNAs interacted with a reported set of myometrial Preterm Initiator genes. RNAs were confirmed by quantitative reverse transcription polymerase chain reaction and tested in a second group of 40 women: 20 with sPTB up to 32weeks (mean gestation 26.5weeks, standard deviation ±2.6weeks), 20 with spontaneous term delivery (40.1±0.9weeks) sampled at 16-19+5 weeks of gestation. Identification of PCF RNAs predictive of sPTB up to 32weeks. Two hundred and ninety-seven PCR RNAs were differentially expressed in sPTB up to 32weeks of gestation. Further selection retained 99 RNAs (86 mRNAs and 13 microRNAs) and five of these interacted in silica with seven Preterm Initiator genes. Four of five RNAs were confirmed and tested on the validation group. The expression of each confirmed PCF RNA was significantly higher in sPTB up to 32weeks of gestation. In vitro study of the four mRNAs revealed higher expression in placentas of women with sPTB up to 32weeks and the potential to interfere with myometrial quiescence. The PCF RNA markers are highly associated with sPTB up to 32weeks by 16weeks of gestation. Women destined for spontaneous preterm birth can be identified by 16weeks of gestation with a panel of maternal plasma RNAs.