Background: Nitroglycerin is suggested to improve flap survival based on promising results; however, there are no data on the effectiveness of treatment initiation time. This study aimed to compare the effect of various nitroglycerin treatment initiation times on partial flap survival. Materials and Methods: The study included 50 Sprague-Dawley rats. Modified McFarlane flaps were elevated on the dorsum of each rat. Group A received placebo treatment. Groups B, C, D, and E received topical nitroglycerin 2% starting 1 day before surgery, on the day of surgery, postoperative d 2, and postoperative d 4, respectively. After 7 days, the flap survival rates were calculated. Afterward, the severity and extent of inflammation and ischemia, and the severity of edema were evaluated histologically. Results: The flap survival rate was highest in group B, followed by groups C, D, E, and A. The difference between groups B and C was not significant, whereas the difference between group B and groups A, D, and E was. In addition, the difference between groups A, D, and E was not significant. Histological analysis showed that inflammation was less severe in groups B and C than in groups A, D, and E. Ischemia was the most severe in groups A and D and was the least severe in group C. Conclusion: Topical nitroglycerin treatment increases flap survival when initiated before or on the day of surgery, but has no benefit when initiated on postsurgery d 2 or 4. Preoperative initiation of nitroglycerin treatment positively affects flap survival.