Introduction. Symptoms associated with newly formed gallstones have never been studied in a population unaware of their gallstones. The objective of this population-based cohort study was to determine which debut of abdominal symptoms was associated with newly formed gallstones. Materials and Methods. A cohort study was performed of a random sample from general population of Copenhagen. Participants had ultrasound examinations and answered questionnaires about abdominal symptoms at baseline and two reexaminations over 12 years. Participants were not informed of gallstone status. Inclusion criteria were no gallstones or cholecystectomy at baseline and attending a reexamination. Results. Of 3,785 participants, 2,845 fulfilled inclusion criteria. Changes in overall abdominal pain were not significantly different between incident gallstones or gallstone-free participants. Multiple adjusted logistic regression analyses showed that incident gallstones were significantly associated with debut of abdominal pain with projection, localized in the whole upper abdomen, and of longer duration. No significant associations for functional symptoms were identified. Conclusions. A new onset of abdominal pain with projection, localized in the whole upper abdomen, and of longer duration is associated with newly formed gallstones in participants unaware of gallstone status. Functional symptoms should not be the indication for surgical treatment.