Problem Inflammation and infection play a major role in preterm birth. The purpose of this study was to (i) determine the prevalence and clinical significance of sterile intra‐amniotic inflammation and (ii) examine the relationship between amniotic fluid (AF) concentrations of high mobility group box‐1 (HMGB1) and the interval from amniocentesis to delivery in patients with sterile intra‐amniotic inflammation. Method of study AF samples obtained from 135 women with preterm labor and intact membranes were analyzed using cultivation techniques as well as broad‐range PCR and mass spectrometry (PCR/ESI‐MS). Sterile intra‐amniotic inflammation was defined when patients with negative AF cultures and without evidence of microbial footprints had intra‐amniotic inflammation (AF interleukin‐6 ≥ 2.6 ng/mL). Results (i) The frequency of sterile intra‐amniotic inflammation was significantly greater than that of microbial‐associated intra‐amniotic inflammation 26% (35/135) versus 11% (15/135); (P = 0.005), (ii) patients with sterile intra‐amniotic inflammation delivered at comparable gestational ages had similar rates of acute placental inflammation and adverse neonatal outcomes as patients with microbial‐associated intra‐amniotic inflammation, and (iii) patients with sterile intra‐amniotic inflammation and high AF concentrations of HMGB1 (≥8.55 ng/mL) delivered earlier than those with low AF concentrations of HMGB1 (P = 0.02). Conclusion (i) Sterile intra‐amniotic inflammation is more frequent than microbial‐associated intra‐amniotic inflammation, and (ii) we propose that danger signals participate in sterile intra‐amniotic inflammation in the setting of preterm labor.