Background: Preoperative localization for small invisible and impalpable pulmonary nodules is important in single-port video-assisted thoracoscopic surgery (VATS). Localization of multiple pulmonary nodules during VATS resection remains challenging. The aim of our study is to elucidate the efficacy of preoperative CT-guided methylene blue localization of both single and multiple pulmonary nodules.Methods: Consecutive patients undergoing preoperative CT-guided methylene blue dye localization for lung nodules, followed by VATS resection, were retrospectively analyzed between January 2014 and November 2019. Chi-square tests, Fisher`s exact test and independent T-test were used to compare variables between the groups. Logistic regression was used to identify risk factors for procedure-related complications.Results: A total of 388 patients, including 337 with single nodule and 51 with multiple nodules, were analyzed. The success rate of preoperative CT-guided methylene blue localization for both single and multiple pulmonary nodules were comparable as 98.8% (333/337) vs. 100% (108/108). The procedure time was longer (23.2 ± 9.4 vs. 7.6 ± 4.8 min, p < 0.001) and risk of pneumothorax was higher (47.1 vs. 25.5%, p = 0.002) in the multiple nodule group. The procedure time (OR 1.079; 95% CI = 1.041–1.118; p < 0.001) was an independent risk factor for pneumothorax. Nodule depth (OR 2.829; 95% CI = 1.259–6.356; p = 0.011) was an independent risk factor for pulmonary hemorrhage.Conclusions: Preoperative CT-guided methylene blue localization for both single and multiple pulmonary nodules is safe, feasible, and effective.