Background: This study aimed to investigate the implication of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) polymorphism on the prognosis of anlotinib monotherapy among patients with treatment-refractory advanced nonsmall cell lung cancer (NSCLC). Methods: Designed as a retrospective study, this study included a total of 129 patients with treatment-refractory advanced NSCLC who were administered with anlotinib monotherapy. The efficacy of the patients was assessed regularly. The prognosis was performed and adverse reactions during anlotinib administration were collected. Available and appropriate biological specimens of the 129 patients were collected to perform VEGFR2 polymorphism analysis and VEGFR2 gene mRNA expression analysis accordingly. Association analysis between genotype status of VEGFR2 polymorphism and other variables was implemented in univariate and multivariate analysis. Results: Efficacy data indicated that the objective response rate (ORR) and disease control rate (DCR) of the 129 patients with NSCLC who received anlotinib monotherapy was 9.3% (95% CI: 4.9%-15.7%) and 78.3% (95%CI: 70.2%-85.1%), respectively. Additionally, prognostic data suggested that the median progression-free survival (PFS) and overall survival (OS) of the 129 patients with NSCLC were 4.1 months (95%CI: 2.84-5.36) and 10.1 months (95%CI: 8.58-11.62), respectively. Furthermore, polymorphism analysis indicated that polymorphism of 4397T>C in VEGFR2 was of clinical significance in the exploratory analysis, which exhibited that the median PFS of patients with TC/CC and TT genotype of 4397T>C polymorphism were 2.8 and 5.0 months, respectively (P = .009). Additionally, patients with TT genotype conferred a superior OS compared with those with TC/CC genotype (median OS: 11.5 vs 7.3 months, P = .016). Interestingly, mRNA expression of the VEGFR2 gene suggested that mRNA expression of VEGFR2 in PBMC specimens of patients with TC/CC genotype was significantly higher than that of patients with TT genotype (P < .001). Conclusion: Anlotinib monotherapy exhibited potential efficacy for patients with treatment-refractory advanced NSCLC. VEGFR2 polymorphism 4397T>C might be used as a promising biomarker to predict the survival of patients with NSCLC who received anlotinib administration.