Finding evidence of anisakidosis requires invasive methods. We have developed a serological assay based on the detection of an immunoglobulin E (IgE) specifically directed against Ani s 1 protein, a major parasite allergen of Anisakis simplex, which has shown a high level of accuracy in the diagnosis of anisakidosis. We used this tool to determine the prevalence of anti-Ani s 1 IgE in dyspeptic patients and to investigate if its seropositivity could be related to epidemiological factors other than raw fish consumption. A total of 174 dyspeptic patients who submitted to upper digestive tract endoscopy were studied. Specific IgE against Ani s 1 was determined by immunoblotting. Quantitative information on smoking, alcohol consumption, and fish consumption as well as a history of gastric surgery was recorded. Twenty-four (13.8%) patients were seropositive for Ani s 1 protein. The seroprevalence of anti-Ani s 1 IgE increased with age in patients who were less than 62 years old (P = 0.047). Seropositivity to Ani s 1 was associated with the consumption of fish in vinegar (P < 0.001), raw fish (P = 0.001), and smoked fish (P = 0.007). There was no relationship between anti-Ani s 1 IgE seropositivity and the number of cigarettes smoked (P = 0.098) or alcohol intake (P = 0.179). Five patients had undergone previous gastric surgery, and three of those patients were seropositive for Ani s 1 (P = 0.019). In multivariate analysis, the consumption of fish in vinegar (P = 0.006), raw fish (P = 0.017), and smoked fish (P = 0.002) and a history of gastric surgery (P = 0.005) were independent factors associated with anti-Ani s 1 IgE detection. In conclusion, at present, anisakidosis might frequently be underdiagnosed, and it might have a clinical role in patients with upper dyspeptic symptoms. Uncooked-fish ingestion and previous gastric surgery were associated with seropositivity for Ani s 1 protein.