Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting， some patients develop chronic infections， such as reactive arthritis， glomerulonephritis， or myocarditis， which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria， chronic infections can only be identified by serological tests. At this point， a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen's strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins， MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis， respectively). Here， we show that Y. enterocolitica and Y. pseudotuberculosis， cultured under the conditions that mimic the natural rout of infection， express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further， we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens， and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.