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The aim of this study was to examine the association between transient interface fluid (TIF) and textural interface opacity (TIO) after Descemet stripping automated endothelial keratoplasty (DSAEK) surgery using intraoperative optical coherence tomography (iOCT) in the PIONEER (Prospective Intraoperative and Perioperative Ophthalmic ImagiNg with Optical CoherEncE TomogRaphy) study.All consecutive eyes that underwent DSAEK between October 2011 and September 2013 from the PIONEER intraoperative and perioperative OCT study were included. iOCT images were captured after lenticule apposition with complete air fill and after air-fluid exchange. Postoperative day 1 OCT images were obtained. Outcome variables included the presence of TIO at the graft–host junction and the presence of intraoperative and postoperative interface fluid on OCT.Seventy-six eyes from 69 patients who underwent DSAEK with iOCT were included. The mean age of the patients was 71 years (range 31–90). The 2 most common indications for surgery were Fuchs dystrophy (63%) and pseudophakic bullous keratopathy (24%). In 18 of 76 (23.7%) eyes, TIF was visible on iOCT post air-fluid exchange. Of these eyes, 14 developed TIO. TIO was observed in 18 of 76 (23.7%) eyes. TIF on iOCT was associated with a significantly higher rate of postoperative TIO (odds ratio = 47.25; P < 0.0001). Sixteen of the 18 eyes that had TIF on iOCT had had resolution on the postoperative day 1 OCT. There was no significant difference in the mean graft thickness between eyes with TIF on iOCT and those without (P = 0.58).Eyes with TIF on iOCT are more likely to develop TIO in the postoperative period. It is believed that the process of gap closure results in TIO, possibly secondary to precipitated solutes, retained viscoelastic, or lamellar irregularities caused by delayed adhesion or uneven matching of lamellar fibrils.