Purpose: To determine factors associated with early flap misalignment following microkeratome-assisted laser in situ keratomileusis. Materials and Methods: This retrospective study included the right eyes of consecutive patients who underwent laser in situ keratomileusis procedure between 2005 and 2016 at Care-Vision Laser Centers, Tel-Aviv, Israel. Patients were divided into two groups according to whether or not they subsequently developed early flap misalignment. Results: A total of 14,582 eyes (mean age of patients: 32.4 ± 10.3 years) were included. Post-laser in situ keratomileusis early flap misalignment developed in 158 eyes (1.1%). Misalignment was more frequent during the spring (32.3% vs 22.8%, p = 0.003) and in a higher operating room temperature (23.34 ± 1.06 vs 22.98 ± 1.26, p < 0.001). In addition, in the misalignment group, there was a higher rate with the of use of the a Moria M2 microkeratome (rather than sub-Bowman’s keratomileusis microkeratome) head (55.2% vs 40.5%, respectively, p < 0.001). In a multivariable analysis adjusted for surgeon and year of surgery, high operating room temperature (odds ratio = 1.22, p = 0.006), treatment zone of 9.0 mm (as opposed to smaller treatment zones, odds ratio = 1.54, p = 0.04), and springtime (odds ratio = 1.58, p = 0.02) were associated with flap misalignment. There was a significant difference in misalignment rates between surgeons (p = 0.02). Conclusion: This study found that larger treatment zones, higher operating room temperature, operating during the spring, and the use of Moria M2 microkeratome were associated with increased flap misalignment rates. The association with operating room temperature and seasonal variation is of interest and merits further research.