Background: Recently, the evaluation of circulating tumor DNA (ctDNA) in pancreatic cancer (PC) patients has gained attention for detection of mutation profile. However, the concordance between tissue next-generation sequencing (NGS) and ctDNA NGS remains unclear. Additionally, the dynamics of genomic alterations during treatment or disease progression are not well understood. Methods: In this prospective cohort study conducted at Seoul National University Hospital from Jan 2022, tissue samples from endoscopic ultrasound-guided fine needle biopsies and blood samples were collected from PC patients. Genomic DNA from the treatment naïve primary tissue and ctDNA from the blood obtained before any treatment and during follow-up with anti-cancer treatment were obtained. Genomic alternation was analyzed based on targeted NGS based panel deep sequencing including 41 genes. Results: The concordance of genomic alteration profiles between tissue NGS and ctDNA NGS was evaluated in 55 patients, and the mutation detection rate of patients who were diagnosed as stage I or II disease were 100% (14 of 14) in tissue NGS and 86% (12 of 14) in ctDNA NGS. That of patients who were diagnosed as stage III or IV disease was 92.7% in tissue NGS (38 of 41) and 90% (37 of 41) in blood ctDNA NGS. Among the study patients, the longitudinal changes of genomic mutation profile were evaluated in the patients at the first treatment response assessment, and patients with progressive disease showed significantly greater amounts of mutations including newly detected Tier1/2 variants than baseline and the previous response assessment. Conclusions: In conclusion, there is comparable mutation detection between tissue genomic DNA and blood ctDNA. The significant increase in ctDNA mutations during disease progression suggests their association with immune evasion and cancer evolution. Further studies utilizing our prospective cohort are needed to explore clinical implications of these results.