The Gram-negative bacteria, Salmonella, cause a broad spectrum of clinical diseases in humans, ranging from asymptomatic carriage to life-threatening sepsis. We have designed an experimental model to study the contribution of genetic factors to the persistence of Salmonella Enteritidis during the late phase of infection in 129S6/SvEvTac and C57BL/6J mice. C57BL/6J mice cleared the bacteria from their reticuloendothelial system within a period of 42 days, whereas the 129S6 mice still presented a high bacterial load. Using this model, we have identified ten Salmonella Enteritidis susceptibility loci (Ses1, Ses1.1, and Ses3-Ses10) associated with bacterial persistence in target organs of 129S6/SvEvTac mice using a two-locus epistasis QTL linkage mapping approach. Significant statistical interactions were detected between Ses1 on chromosome 1 and Ses5 on chromosome 7 and between Ses1 and Ses4 on chromosome X. In this study, we functionally validated the genetic architecture of Salmonella persistence in 129S6 mice using single- (129S6.B6-Ses1.2 that combines Ses1 and Ses1.1 loci, 129S6.B6-Ses4, and 129S6.B6-Ses5) and double-congenic mice (129S6.B6-Ses1.2/Ses4 and 129S6.B6-Ses1.2/Ses5). These experiments demonstrate functional interactions between Ses1.2 and Ses4 or Ses5 that improve Salmonella Enteritidis clearance, validating the critical role played by gene-gene interactions in the contribution to bacterial clearance heritability. Improved bacterial clearance in double-congenic mice could be explained by the impact of Ses4 and Ses5 in combination with Ses1.2 on TH polarization since a TH2 bias (decreased Ifng and increased Il4 mRNA levels and reduced IgG2a immunoglobulins in the serum) was observed in 129S6.B6-Ses1.2/Ses5 mice and a TH17 (high Il17 expression) bias in 129S6.B6-Ses1.2/Ses4.