Purpose To report the incidence and associated factors for the development of vitreomacular interface abnormality (VMIA) in patients with diabetic macular edema (DME) who received intravitreal injection (IVI) of anti-VEGF (Bevacizumab and Ranibizumab) treatment. Methods A retrospective observational study. Patients with DME followed at least 6 months were reviewed. Baseline bestcorrected visual acuity (BCVA), central retinal thickness (CRT) and final BCVA, CRT in eyes with and without VMIA were compared. Multiple logistic regression was also used to investigate the risk factors of VMIA formation in patients with DME treated by anti-VEGF. Results A total of 201 eyes in 142 patients met the inclusion criteria of the study. VMIA developed in 44 eyes (21.89%) of patients during a mean follow-up period of 40.84 months. The estimated mean incidence of VMIA formation was 6.43% per year. Poor baseline BCVA was found to be a risk factor for VMIA development (P = 0.001, odds ratio = 5.299, 95% confidence interval: 1.972 to 14.238). There was no difference between eyes with and without VMIA formation in improving BCVA (P = 0.557) and lowering the macular edema (eyes without VMIA formation: − 107.72 ± 171.91 μm; eyes with VMIA formation: − 155.02 ± 212.27 μm, P = 0.133). Conclusions This study revealed the incidence of VMIA formation in IVI anti-VEGF treated DME eyes was 6.43%.Poor baseline BCVA was found to be a risk factor for VMIA formation. Both eyes with and without VMIA development had favorable response to anti-VEGF treatment.