The Eurasian lineages of swine influenza viruses are different genetically from classical swine H1N1 influenza viruses and comprise avian-like H1N1 and human-like H1N2 and H3N2 subtypes. Although sporadic isolation of such viruses from human specimens has been reported, the prevalence of human infections is not known. In the present study, the seroprevalence against Eurasian swine influenza viruses was investigated. Sera were collected in Thuringia, Germany, from December 2007 to April 2009. The study group comprised 118 professionals with occupational exposure to pigs (50 pig slaughterers/meat inspectors, 46 pig farmers, 22 veterinarians caring for pig herds). The control group included 118 age- and gender-matched blood donors from Thuringia. As a result, 18 sera of the study group were identified with raised hemagglutination-inhibition titers against a panel of nine swine influenza viruses (three strains/ subtype). For 17/18 sera this finding was confirmed in the neutralization assay. For 11/18 sera the raise of titers was significant, that is, a fourfold increase of hemagglutination-inhibition titers was observed. No gender-specific bias of the high titer sera was observed. Twelve sera of the control group showed increased hemagglutination-inhibition titers against swine influenza viruses. Hemagglutination-inhibition titers of 2/12 control sera were raised fourfold but did not exhibit a significant increase of neutralization titers. All increased hemagglutination-inhibition titers of the control group may be explained by cross-reactivity with seasonal influenza virus strains, as all these sera also reacted with human strains. J. Med. Virol. 82:1617–1625, 2010. © 2010 Wiley-Liss, Inc.