Biomarkers for early diagnosis of patients with pancreatic cancer (PC) are needed. Our aim was to identify panels of miRNAs in serum in combination with CA 19-9 for use in the diagnosis of PC. Four hundred seventeen patients with PC were included prospectively from Denmark (n 5 306) and Germany (n 5 111). Controls included 59 patients with chronic pancreatitis (CP) and 248 healthy subjects (HS). MiRNAs were analyzed in pretreatment serum samples from 3 cohorts: discovery cohort (754 human miRNAs, TaqMan V R Human MicroRNA assay, Applied Biosystem; PC n 5 133, controls n 5 72); training cohort (34 miRNAs, real-time qPCR using the Fluidigm BioMark TM System; PC n 5 198, controls n 5 184); validation cohort (13 miRNAs, real-time qPCR using the Fluidigm BioMark TM System; PC n 5 86, controls n 5 51). We found that 34 miRNAs in serum from PC patients in the discovery cohort were expressed differently than in controls. These miRNAs were tested in the training cohort, and four diagnostic panels were constructed that included 5 or 12 miRNAs (miR-16, 218a, 220a, 224, 225, 227a, 229c, 230a.5p, 2191, 2323.3p, 2345 and 2483.5p). In conclusion, we identified four diagnostic panels based on 5 or 12 miRNAs in serum that could distinguish patients with PC from HS and CP.In the Western world, 280,000 subjects develop pancreatic cancer (PC) each year, and the incidence is increasing. The prognosis for these patients has improved only slightly during the last decade, and PC is the fourth-leading cause of cancer-related deaths in the US (42.000) and Europe (75.000). 1-3 <20% of patients with PC can be operated on with curative intent, because most patients have locally advanced or metastatic PC at the time of diagnosis. 4 Chemotherapy has little effect, 4 and only 20% are alive after 1 year and just 7% after 5 years.