To correlate the degree of anterior chamber (AC) inflammation and corneal thickness evaluated by anterior segment optical coherence tomography (ASOCT) with corneal graft rejection status and to explore the value of ASOCT in assisting the diagnosis or prediction of graft rejection using a rat penetrating keratoplasty (PK) model. A total of 40 PKs were performed using Fisher rats (allogeneic groups) and Lewis rats (syngeneic group) as donors and Lewis rats as recipients: isograft control group (n=10), allograft untreated group (n=10), and allograft with 1% prednisolone acetate treatment group (n=20). All the grafts were evaluated for 28 days by a scoring rejection index (RI) to assess the graft opacity, edema, and neovascularization using slit lamp biomicroscopy. The AC inflammation and corneal graft thickness were assessed using ASOCT. All the allogeneic control grafts and four of the 20 allogeneic steroid-treated grafts developed rejection episodes. In the allogeneic treated group, the rejected grafts had a significantly higher mean AC inflammation grade at 1 week (grade 3.25±0.49 vs. 1.83±0.36, P<0.001), significantly thicker central graft thickness at 2 weeks (455.25±42.42 μm vs. 381.247±12.51 μm, P=0.047), and a significantly higher RI at 4 weeks (7.75±0.63 vs. 4.60±0.13, P<0.001) compared to the nonrejected grafts. Eyes with ≥grade 3 AC inflammation at 1 week, or with ≥400 μm central graft thickness at 2 weeks, were significantly associated with graft rejection (odds ratio [OR], 15.15, P=0.009, and OR, 9.75, P=0.014, respectively). The use of ASOCT to evaluate AC inflammation and corneal thickness aids in the early evaluation and diagnosis of graft rejection in animal models. Early increased AC inflammation was an early predictor of graft failure prior to definitive clinical evaluation.