Introduction: Early-stage non-small cell lung cancer (NSCLC) has a high recurrence rate despite proper management, including curative surgery. Circulating tumor cells (CTCs) are believed to play a key role in the distant metastasis of lung cancer. Immunofluorescent imaging studies of CTCs have revealed that they are associated with the prognosis of NSCLC. However, the mutational profiling of CTCs from early-stage NSCLC has not been extensively explored. We hypothesized that CTCs could be detected by mutational analysis using panel sequencing and would have distinct mutations associated with distant metastasis compared to those of primary tumor tissue and cell-free DNA (cfDNA) in early-stage NSCLC. Thus, this study examined the DNA from CTCs using targeted panel sequencing to identify mutations and compared them with mutations found in primary tumor tissue and cfDNA in patients with resectable early-stage NSCLC. Methods: Overall, 50 patients with resectable lung cancer were prospectively enrolled from September to December 2023. Matched whole blood samples and primary tumor tissues were collected during curative surgery. Buffy coat and plasma for each CTCs and cfDNA were extracted by centrifuge. Then, targeted panel sequencing was performed on DNA from primary tumor tissues, cfDNA and CTCs. Results: In this study, 37 patients (74%) had adenocarcinoma, and 33 (66%) were classified as having pathologic stage 1 disease. Mutated genes were detected in all (100%), 49 (98%) and 34 patients (68%) for primary tumor tissue, cfDNA and CTCs from panel sequencing, respectively. The most frequently mutated gene in CTCs was MSH6 while EGFR and CDH1 were the most common in primary tumor tissue and cfDNA, respectively. The top 10 mutated genes differed significantly across the sample types. Among primary tumor tissue samples, mutated genes differed by stage and histologic type; these findings were not observed in CTCs. CTCs predominantly displayed mutations in tumor suppressor genes, whereas primary tumor tissues exhibited mutations in both oncogenes and tumor suppressor genes. Conclusion: CTCs exhibited unique mutations, showing low concordance with mutations found in primary tumor tissue and cfDNA. CTCs may possess specific mutations independent from those of primary tumor tissue and cfDNA in early-stage NSCLC.