<p>The vast majority of patients with pancreatic cancer present with unresectable disease and precision medicine is lagging behind. Circulating tumor DNA (ctDNA) has emerged as a promising tool, both as a proxy for tumor burden and for capturing tumor heterogeneity, but optimal gene panels and prognostic cutoffs remain to be determined. Herein, we applied ultra-deep ctDNA sequencing using a customized panel targeting 23 genes and six frequently altered chromosomes on plasma samples obtained before, during and after chemotherapy from 60 patients enrolled in a prospective clinical study. At baseline, positive versus negative ctDNA was not prognostic, neither in the adjuvant nor in the palliative setting, but in palliative patients, an independent prognostic cutoff could be calculated from the absolute number of mutated DNA molecules. Median overall survival was 3.7 months in the ctDNA^high compared to 11.9 months in the ctDNA^low group (<i>p</i> &lt; 0.0001), and the cutoff remained prognostic at one and three months. Moreover, relevant genetic alterations were highly concordant in ctDNA and paired tumor tissue. These findings demonstrate the potential clinical utility of a customized and focused gene panel for prognostication and target identification over time in patients with newly diagnosed pancreatic cancer, in particular in the palliative setting.</p><p>ClinicalTrials.gov number: NCT03724994.</p>